Pyrrolobenzodiazepine antibody conjugates

ABSTRACT

The present disclosure relates generally to antibody-drug conjugates comprising pyrrolo[2, 1-c][1, 4]benzodiazepine (PBD) drug moieties. The present disclosure also relates to methods of using these conjugates, e.g., as therapeutics and/or diagnostics.

RELATED APPLICATIONS

This application is a U.S. National Phase application, filed underU.S.C. § 371, of International Application No. PCT/US2018/062505, filedNov. 27, 2018, which claims priority to, and the benefit of, U.S.Provisional Application Nos. 62/590,893, filed Nov. 27, 2017,62/653,757, filed Apr. 6, 2018, and 62/701,090, filed Jul. 20, 2018,under 35 U.S.C. § 119(e). The content of each of these applications arehereby incorporated by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “MRSN-022N01US_SeqList.txt”, whichwas created on May 27, 2020 and is 3 KB in size, are hereby incorporatedby reference in their entirety.

BACKGROUND

The pyrrolo[2, 1-c][1, 4]benzodiazepines (PBDs) area family of naturallyoccurring, monofunctional DNA alkylating antitumor antibiotics, whichincludes anthramycin, DC-81, tomaymycin, and sibiromycin. Thesecompounds bind exclusively to the exocyclic N2 of guanine in the minorgroove and span 3 base pairs in a sequence specific manner (5′PuGPu).The first PBD antitumor antibiotic, anthramycin, was discovered in 1965(Leimgruber et al., 1965. J. Am. Chem. Soc., 87, 5793-5795; andLeimgruber et al., 1965 J. Am. Chem. Soc., 87, 5791-5793). Since then, anumber of naturally occurring PBDs and variety of analogues have beenreported.

PBDs have the general structure:

The PBDs differ in the number, type and position of substituents, inboth their aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)) or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position which is the electrophilic center responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, 1975 In Antibiotics III. Springer-Verlag, New York, pp.3-11; and Hurley and Needham-VanDevanter, 1986 Acc. Chem. Res., 19,230-237). Their ability to form an adduct in the minor groove enablesthem to interfere with DNA processing, hence their use as antitumoragents.

The first PBD to enter the clinic, SJG-136 (NSC 694501) is a potentcytotoxic agent that causes DNA inter-strand crosslinks (S. G Gregson etal., 2001, J. Med. Chem., 44: 737-748; M. C. Alley et al., 2004, CancerRes., 64: 6700-6706; J. A. Hartley et al., 2004, Cancer Res., 64:6693-6699; C. Martin et al., 2005, Biochemistry., 44: 4135-4147; S.Arnould et al., 2006, Mol. Cancer Ther., 5: 1602-1509). Results from aPhase I clinical evaluation of SJG-136 revealed that this drug was toxicat extremely low doses (maximum tolerated dose of 45 μg/m, and severaladverse effects were noted, including vascular leak syndrome, peripheraledema, liver toxicity and fatigue. DNA damage was noted at all doses incirculating lymphocytes.

Accordingly, there exists a need for more selective and efficaciousdrugs that can deliver critical DNA damage with minimal side effectscontinues.

SUMMARY

The present disclosure provides, inter alia, an antibody-drug conjugate(ADC) of Formula (I):PBRM-[L^(C)-D]_(d15)   (I)or a pharmaceutically acceptable salt or solvate thereof, wherein:

PBRM denotes a protein based recognition-molecule;

L^(C) is a linker unit connecting the PBRM to D;

D is a PBD drug moiety; and

d₁₅ is an integer from about 1 to about 20.

In some embodiments, the conjugate is of Formula (II):

or a pharmaceutically acceptable salt or solvate thereof, wherein:

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM;

M^(P) is a Stretcher unit;

a₁ is an integer from 0 to 1;

M^(A) comprises a peptide moiety that contains at least two amino acids;

T′ is a hydrophilic group and the

between T′ and M^(A) denotes direct or indirect attachment of T′ andM^(A);

each occurrence of L^(D) is independently a divalent linker moietyconnecting D to M^(A) and comprises at least one cleavable bond suchthat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and

d₁₃ is an integer from 1 to 20.

In some embodiments, d₁₃ is an integer from 2 to 14, from 2 to 12, from2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, from 4 to 10, from 4 to8, from 4 to 6, from 6 to 14, from 6 to 12, from 6 to 10, from 6 to 8,from 8 to 14, from 8 to 12, or from 8 to 10.

In some embodiments, d₁₃ is 3 to 5

In some embodiments, d₁₃ is 4 or 5.

In some embodiments, L^(P), when not connected to PBRM, comprises aterminal group W^(P), in which each W^(P) independently is:

wherein

R^(1K) is a leaving group;

R^(1A) is a sulfur protecting group;

ring A is cycloalkyl or heterocycloalkyl;

ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R^(1J) is hydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety;

R^(2J) is hydrogen or an aliphatic, aryl, heteroaliphatic, orcarbocyclic moiety;

R^(3J) is C₁₋₆ alkyl;

Z₁, Z₂, Z₃ and Z₇ are each independently a carbon or nitrogen atom;

R^(4j) is hydrogen, halogen, OR, —NO₂, —CN, —S(O)₂R, C₁₋₂₄ alkyl (e.g.,C₁₋₆alkyl), or 6-24 membered aryl or heteroaryl, wherein the C₁₋₂₄ alkyl(e.g., C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl, is optionallysubstituted with one or more aryl or heteroaryl; or two R^(4j) togetherform an annelated cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; Ris hydrogen, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl

R is hydrogen or aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety;

R^(5j) is C(R^(4j))₂, O, S or NR; and

z₁ is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some embodiments, each R^(1K) is halo or RC(O)O— in which R ishydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety.

In some embodiments, each R^(1A) independently is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogenor an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkylmoiety.

In some embodiments, L^(P), when not connected to PBRM is

In some embodiments, M^(P), when present, is —(Z₄)—[(Z₅)—(Z₆)]_(z)—,with Z₄ connected to L^(P′) or L^(P) and Z₆ connected to L^(M); in which

z is 1, 2, or 3;

Z₄ is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to Z₅ or Z₆ when present or to M^(A) when Z₅ and Z₆ are bothabsent

b₁ is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

R₁₇ is C₁₋₁₀ alkylene, C₁₋₁₀ heteroalkylene, C₃₋₈ cycloalkylene, O—(C₁₋₈alkylene, arylene, —C₁₋₁₀ alkylene-arylene-, -arylene-C₁₋₁₀ alkylene-,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-, —(C₃₋₈ cycloalkylene —C₁₋₁₀alkylene-, 4 to 14-membered heterocycloalkylene, —C₁₋₁₀ alkylene-(4 to14-membered heterocycloalkylene)-, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-, —C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀heteroalkylene-C(═O)—, —C₃₋₈ cycloalkylene-C(═O)—, —O—(C₁₋₈alkyl)-C(═O)—, -arylene-C(═O)—, —C₁₋₁₀ alkylene-arylene-C(═O)—, -arylene—C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-C(═O)—,—(C₃₋₈ cycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, -4 to 14-memberedheterocycloalkylene-C(═O)—, —C₁₋₁₀ alkylene-(4 to 14-memberedheterocycloalkylene)-C(═O)—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-NH—, —C₁₋₁₀heteroalkylene-NH—, —C₃₋₈ cycloalkylene-NH—, —O—(C₁₋₈ alkyl)-NH—,-arylene-NH—, —C₁₋₁₀ alkylene-arylene-NH—, -arylene-C₁₋₁₀ alkylene-NH—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-NH—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-NH—, -4 to 14-membered heterocycloalkylene-NH—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-NH—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-NH—, —C₁₋₁₀ alkylene-S—, —C₁₋₁₀heteroalkylene-S—, —C₃₋₈ cycloalkylene-S—, —O—C₁₋₈ alkyl)-S—,-arylene-S—, —C₁₋₁₀ alkylene-arylene-S—, -arylene-C₁₋₁₀ alkylene-S—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-S—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-S—, -4 to 14-membered heterocycloalkylene-S—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-S—, or -(4 to14-membered heterocycloalkylene)-C₁-C₁₀ alkylene-S—;

each Z₅ independently is absent, R₅₇—R₁₇ or a polyether unit,

each R₅₇ independently is a bond, NR₂₃, S or O;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl; and

each Z₆ independently is absent, —C₁₋₁₀ alkyl-R₃—, —C₁₋₁₀ alkyl-NR₅—,—C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-S— or —(C₁₋₁₀alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—;

each R₃ independently is —C(O)—NR₅— or —NR₅—C(O)—;

each R₅ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, COOH, or COO—C₁₋₆ alkyl; and

g₁ is an integer from 1 to 4.

In some embodiments, Z₄ is

in which b₁ is 1 or 4.

In some embodiments, Z₄ is

in which b₁ is 1 or 4.

In some embodiments, Z₄ is

In some embodiments, Z₄ is

In some embodiments, each Z₅ independently is a polyalkylene glycol(PAO).

In some embodiments, M^(P), when present, is

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M);

R₃, R₅, R₁₇, and R₂₃ are as defined herein;

R₄ is a bond or —NR₅—(CR₂₀R₂₁)—C(O)—;

each R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

each b₁ independently is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

each f₁ independently is an integer from 1 to 6; and

g₂ is an integer from 1 to 4.

In some embodiments, M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M).

In some embodiments, M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to M^(A).

In some embodiments, M^(A) comprises a peptide moiety of at least twoamino acid (AA) units.

In some embodiments, L^(D) comprises a peptide of 1 to 12 amino acids,wherein each amino acid is independently selected from alanine,3-alanine, arginine, aspartic acid, asparagine, histidine, glycine,glutamic acid, glutamine, phenylalanine, lysine, leucine, serine,tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine,methionine, selenocysteine, ornithine, penicillamine, aminoalkanoicacid, aminoalkynoic acid, aminoalkanedioic acid, aminobenzoic acid,amino-heterocyclo-alkanoic acid, heterocyclo-carboxylic acid,citrulline, statine, diaminoalkanoic acid, and derivatives thereof.

In some embodiments, L^(D) comprises β-alanine.

In some embodiments, L^(D) comprises (β-alanine)-(alanine)-(alanine) or(β-alanine)-(valine)-(alanine).

In some embodiments, T′ comprises a polyalcohol or a derivative thereof,a polyether or a derivative thereof, or a combination thereof.

In some embodiments, T′ comprises an amino polyalcohol.

In some embodiments, T′ comprises one or more of the following fragmentsof the formula:

in which

n₁ is an integer from 0 to about 6;

each R₅₈ is independently hydrogen or C₁₋₈ alkyl;

R₆₀ is a bond, a C₁₋₆ alkyl linker, or —CHR₅₉— in which R₅₉ is H, alkyl,cycloalkyl, or arylalkyl;

R₆₁ is CH₂OR₆₂, COOR₆₂, —(CH₂)_(n2)COOR₆₂, or a heterocycloalkylsubstituted with one or more hydroxyl;

R₆₂ is H or C₁₋₈ alkyl; and

n₂ is an integer from 1 to about 5.

In some embodiments, T′ comprises glucamine.

In some embodiments, T′ comprises:

In some embodiments, T′ comprises:

in which

n₄ is an integer from 1 to about 25;

each R₆₃ is independently hydrogen or C₁₋₈ alkyl;

R₆₄ is a bond or a C₁₋₈ alkyl linker;

R₆₅ is H, C₁₋₈ alkyl, —(CH₂)_(n2)COOR₆₂, or —(CH₂)_(n2)COR₆₆;

R₆₂ is H or C₁₋₈ alkyl;

R₆₆ is

and

n₂ is an integer from 1 to about 5.

In some embodiments, T′ comprises polyethylene glycol, e.g.,polyethylene glycol with from about 6 to about 24 PEG subunits,preferably from about 6 to about 12 PEG subunits, or from about 8 toabout 12 PEG subunits.

In some embodiments, T′ comprises:

in which n₄ is an integer from about 2 to about 20, from about 4 toabout 16, from about 6 to about 12, from about 8 to about 12.

In some embodiments, n₄ is 6, 7, 8, 9, 10, 11, or 12.

In some embodiments, n₄ is 8 or 12.

In some embodiments, T′ comprises:

in which n₄ is an integer from about 2 to about 20, from about 4 toabout 16, from about 6 to about 12, or from about 8 to about 12.

In some embodiments, n₄ is 6, 7, 8, 9, 10, 11, or 12.

In some embodiments, n₄ is 8 or 12.

In some embodiments, the conjugate is of Formula (III):PBRM-(A¹ _(a6)-L¹ _(s2)L² _(y1)-D)_(d13)  (III)or pharmaceutically acceptable salt or solvate thereof, wherein:

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

A¹ is a stretcher unit;

a₆ is an integer 1 or 2;

L¹ is a specificity unit;

s₂ is an integer from about 0 to about 12;

L² is a spacer unit;

y1 is an integer from 0 to 2; and

d₁₃ is an integer from about 1 to about 20.

In some embodiments, the conjugate is of any one of Formulae (IIIa) to(IIIf):

or a pharmaceutically acceptable salt or solvate thereof, wherein:

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

A¹ is a stretcher unit linked to the spacer unit L²;

a₆ is an integer 1 or 2;

L¹ is a specificity unit linked to the spacer unit L²;

s₂ is an integer from about 0 to about 12;

s₆ is an integer from about 0 to about 12.

L² is a spacer unit;

y₁ is an integer 0, 1 or 2; and

d₁₃ is an integer from about 1 to about 20.

In some embodiments, the PBD drug moiety (D) is of Formula (IV):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer, wherein:

E″ is a direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment), E, or

in which

denotes direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment) via a functional group of E;

D″ is D′ or

in which

denotes direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment) via a functional group of D′;

R″₇ is a direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment), R₇, or

in which

denotes direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment) via a functional group of R₇;

R″₁₀ is a direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment), R₁₀ or

in which

denotes direct or indirect linkage the PBRM (e.g., antibody or antibodyfragment) via a functional group of R₁₀; and

wherein the PBD drug moiety (D) is directly or indirectly linked to thePBRM (e.g., antibody or antibody fragment) via a functional group of oneof E″, D″, R″₇, and R″₁₀.

In some embodiments, E″ is a direct or indirect linkage to L^(C), E, or

in which

denotes direct or indirect linkage to L^(C) via a functional group of E.

In some embodiments, E″ is a direct or indirect linkage to L^(D), E, or

in which

denotes direct or indirect linkage to L^(D) via a functional group of E.

In some embodiments, D″ is D′ or

in which

denotes direct or indirect linkage to L^(C) via a functional group ofD′.

In some embodiments, D″ is D′ or

in which

denotes direct or indirect linkage to L^(D) via a functional group ofD′.

In some embodiments, R″₇ is a director indirect linkage to L^(C), R₇ or

in which

denotes direct or indirect linkage to L^(C) via a functional group ofR₇.

In some embodiments, R″₇ is a direct or indirect linkage to L^(D), R₇ or

in which

denotes direct or indirect linkage to L^(D) via a functional group ofR₇.

In some embodiments, R″₁₀ is a director indirect linkage to L^(C), R₁₀,or

in which

denotes direct or indirect linkage L^(C) via a functional group of R₁₀.

In some embodiments, R″₁₀ is a direct or indirect linkage to L^(D), R₁₀,or

in which

denotes direct or indirect linkage L^(C) via a functional group of R₁₀.

In some embodiments, E″ is a direct or indirect linkage to the PBRM; D″is D′; R″₇ is R₇ and R″₁₀ is R₁₀.

In some embodiments, E″ is a director indirect linkage to L^(C); D″ isD′; R″₇ is R₇ and R″₁₀ is R₁₀.

In some embodiments, E″ is a direct or indirect linkage to L^(D); D″ isD′; R″₇ is R₇ and R″₁₀ is R₁₀.

In some embodiments, E″ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofE; D″ is D′; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, E″ is

in which

denotes direct or indirect linkage to L^(C) via a functional group of E;D″ is D′; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, E″ is

in which

denotes direct or indirect linkage to L^(D) via a functional group of E;D″ is D′; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, D″ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofD; E″ is E; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, D″ is

in which

denotes direct or indirect linkage to L^(C) via a functional group of D;E″ is E; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, D″ is

in which

denotes direct or indirect linkage to L^(D) via a functional group of D;E″ is E; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is a direct or indirect linkage to the PBRM; E″is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is a direct or indirect linkage to L^(C); E″ isE; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is a direct or indirect linkage to L^(D); E″ isE; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofR₇; E″ is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is

in which

denotes direct or indirect linkage to L^(C) via a functional group ofR₇; E″ is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is

in which

denotes direct or indirect linkage to L^(D) via a functional group ofR₇; E″ is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₁₀ is a director indirect linkage to the PBRM; E″is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is a director indirect linkage to L^(C); E″ isE, D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is a direct or indirect linkage to L^(D); E″is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofR₁₀; E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is

in which

denotes direct or indirect linkage to L^(C) via a functional group ofR₁₀; E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is

in which

denotes direct or indirect linkage to L^(D) via a functional group ofR₁₀, E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, D′ is D1. D2, D3, or D4:

wherein the dotted line between C₂ and C₃ or between C₂ and C₁ in D1 orthe dotted line in D4 indicates the presence of a single or double bond;and

m is 0, 1 or 2;

when D′ is D1, the dotted line between C₂ and C₃ is a double bond, and mis 1, then R₁ is:

(i) C₆₋₁₀ aryl group, optionally substituted by one or more substituentsselected from —OH, halo, —NO₂, —CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂,—OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, a polyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a), 3- to14-membered heterocycloalkyl, 5- to 12-membered heteroaryl, bis-oxy-C₁₋₃alkylene, —NR₃R₄, —S(═O)₂R₁₂, —S(═O)₂NR₁₃R₁₄, —SR₁₂, —SO_(x)M,—OSO_(x)M, —NR₉COR₁₉, —NH(C═NH)NH₂;

(ii) C₁₋₈ alkyl;

(iii) C₃₋₆ cycloalkyl;

or

(viii) halo;

when D′ is D1, the dotted line between C2 and C3 is a single bond, and mis 1, then R₁ is:

(i) —OH, ═O, ═CH₂, —CN, —R₂, —OR₂, halo, ═CH—R₆, ═C(R₆)₂, —O—SO₂R₂,—CO₂R₂, —COR₂, —CHO, or —COOH; or

when D′ is D1 and m is 2, then each R₁ independently is halo and eitherboth R₁ are attached to the same carbon atom or one is attached to C₂and the other is attached to C₃;

T is C₁₋₁₀ alkylene linker;

A is

wherein the —NH group of A is connected to the —C(O)-T- moiety ofFormula (I) and the C═O moiety of A is connected to E; and each

independently is

E is E1, E2, E3, E4, —OH, —NH—(C₁₋₆ alkylene)-R_(13a), —O—(CH₂)₃—NH₂,—O—CH(CH₃)—(CH₂)₂—NH₂, or —NH—(CH₂)₃—O—C(═O)—CH(CH₃)—NH₂:

wherein the dotted line in E1 or E4 indicates the presence of a singleor double bond;

each occurrence of R₂ and R₃ independently is an optionally substitutedC₁₋₈ alkyl, optionally substituted C₂₋₈ alkenyl, optionally substitutedC₂₋₈ alkynyl, optionally substituted C₃₋₈ cycloalkyl, optionallysubstituted 3- to 20-membered heterocycloalkyl, optionally substitutedC₆₋₂₀ aryl or optionally substituted 5- to 20-membered heteroaryl, and,optionally in relation to the group NR₂R₃, R₂ and R₃ together with thenitrogen atom to which they are attached form an optionally substituted4-, 5-, 6- or 7-membered heterocycloalkyl or an optionally substituted5- or 6-membered heteroaryl;

R₄, R₅ and R₇ are each independently —H, —R₂, —OH, —OR₂, —SH, —SR₂,—NH₂, —NHR₂, —NR₂R₃, —NO₂, —SnMe₃, halo or a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a); or R₄ and R₇ together form bis-oxy-C₁₋₃ alkylene;

each R₆ independently is —H, —R₂, —C₂R₂, —COR₂, —CHO, —CO₂H, or halo;

each R₈ independently is —OH, halo, —NO₂, —CN, —N₃, —OR₂, —COOH, —COOR₂,—COR₂, —OCONR₁₃R₁₄, —CONR₁₃R₁₄, —CO—NH—(C₁₋₆ alkylene)-R_(13a),C₁₋₁₀alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, apolyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a), 3- to 14-memberedheterocycloalkyl, 5- to 12-membered heteroaryl, —S(═O)₂R₂,—S(═O)₂NR₁₃R₁₄, —SR₁₂, —SO_(x)M, —OSO_(x)M, —NR₉COR₁₉, —NH(C═NH)NH₂,—R₂₀—R₂₁—NR₁₃R₁₄, —R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃;

each R₉ independently is C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl;

R¹⁰ is —H or a nitrogen protecting group,

R¹¹ is -QR^(Q) or —SO_(x)M;

or R¹⁰ and R¹¹ taken together with the nitrogen atom and carbon atom towhich they are respectively attached, form a N═C double bond;

each R₁₂ independently is C₁₋₇ alkyl, 3- to 20-memberedheterocycloalkyl, 5- to 20-membered heteroaryl, or C₆₋₂₀ aryl

each occurrence of R₁₃ and R₁₄ are each independently H, C₁₋₁₀ alkyl, 3-to 20-membered heterocycloalkyl, 5- to 20-membered heteroaryl, or C₆₋₂₀aryl;

each R_(13a) independently is —OH or —NR₁₃R₁₄;

R₁₅, R₁₆, R₁₇ and R₁₈ are each independently —H, —OH, halo, —NO₂, —CN,—N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3-14 membered heterocycloalkyl, 5- to 12-memberedheteroaryl, —NR₁₃R₁₄, —S(═O)₂R₂, —S(═O)₂NR₁₃R₁₄, —SR₁₂, —SO_(x)M,—OSO_(x)M, —NR₉COR₁₉ or —NH(C═NH)NH₂;

each R₁₉ independently is C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenylor C₂₋₁₀ alkynyl;

each R₂₀ independently is a bond, C₆₋₁₀ arylene, 3-14 memberedheterocycloalkylene or 5- to 12-membered heteroarylene;

each R₂₁ independently is a bond or C₁₋₁₀ alkylene;

R₃₁, R₃₂ and R₃₃ are each independently —H, C₁₋₃ alkyl, C₂₋₃ alkenyl,C₂₋₃ alkynyl or cyclopropyl, wherein the total number of carbon atoms inthe R₁ group is no more than 5;

R₃₄ is —H, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, cyclopropyl, orphenyl wherein the phenyl is optionally substituted by one or more ofhalo, methyl, methoxy, pyridyl or thiophenyl;

one of R_(35a) and R_(35b) is —H and the other is a phenyl groupoptionally substituted with one or more of halo, methyl, methoxy,pyridyl or thiophenyl;

R_(36a), R_(36b), R_(36c) are each independently —H or C₁₋₂ alkyl;

R_(36d) is —OH, —SH, —COOH, —C(O)H, —N═C═, —NHNH₂, —CONHNH₂,

or NHR^(N), wherein R^(N) is —H or C₁₋₄ alkyl;

R_(37a) and R_(37b) are each independently is —H, —F, C₁₋₄ alkyl, C₂₋₃alkenyl, wherein the alkyl and alkenyl groups are optionally substitutedby C₁₋₄ alkyl amido or C₁₋₄ alkyl ester; or when one of R_(37a) andR_(37b) is —H, the other is —CN or a C₁₋₄ alkyl ester;

R₃₈ and R₃₉ are each independently H, R₁₃, ═CH₂, ═CH—(CH₂)_(s1)—CH₃, ═O,(CH₂)_(s1)—OR₁₃, (CH₂)_(s1)—CO₂R₁₃(CH₂)_(s1)—NR₁₃R₁₄, O—(CH₂)₂—NR₁₃R₁₄,NH—C(O)—R₁₃, O—(CH₂)s-NH—C(O)—R₁₃, O—(CH₂)s-C(O)NHR₃,(CH₂)_(s1)OS(═O)₂R₁₃, O—SO₂R₃, (CH₂)_(s1)—C(O)R₁ and(CH₂)_(s1)—C(O)NR₁₃R₁₄;

X₀ is CH₂, NR₆, C═O, BH, SO or SO₂;

Y₀ is O, CH₂, NR₆ or S;

Z₀ is absent or (CH₂)_(n);

each X₁ independently is CR_(b), or N;

each Y₁ independently is CH, NR_(a), O or S;

each Z₁ independently is CH, NR_(a), O or S;

each R_(a) independently is H or C₁₋₄ alkyl;

each R_(b) independently is H, OH, C₁₋₄ alkyl, or C₁₋₄ alkoxyl;

X₂ is CH, CH₂ or N;

X₃ is CH or N;

X₄ is NH, O or S;

X₈ is NH, O or S;

Q is O, S or NH;

when Q is S or NH, then R^(Q) is —H or optionally substituted C₁₋₂alkyl; or

when Q is O, then R^(Q) is —H or optionally substituted C₁₋₂ alkyl,—SO_(x)M, —PO₃M, —(CH₂—CH₂—O)_(n9)CH₃, —(CH₂—CH₂O)_(n1)—CH₂)₂—R₄₀,—C(O)—(CH₂—CH₂₋₀)_(n9)CH₃, —C(O)O—(CH₂—CH₂—O)_(n9)CH₃,—C(O)NH—(CH₂—CH₂—O)_(n9), CH₃,—(CH₂)_(n)—NH—C(O)—CH₂—O—CH₂—C(O)—NH—(CH₂—CH₂—O)_(n9)CH₃,—(CH)_(n)—NH—C(O)—(CH₂)_(n)—(CH₂—CH₂—O)_(n9)CH₃, a sugar moiety,

each M independently is H or a monovalent pharmaceutically acceptablecation;

n is 1, 2 or 3;

n₉ is 1, 2, 3, 4, 5, 6, 8, 12 or 24.

each r independently is an integer from 1 to 200;

s is 1, 2, 3, 4, 5 or 6;

s₁ is 0, 1, 2, 3, 4, 5 or 6;

t is 0, 1, or 2;

R₄₀ is —SO₃H, —COOH, —C(O)NH(CH₂)₂SO₃H or —C(O)NH(CH₂)₂COOH; and

each x independently is 2 or 3.

In some embodiments, the PBD drug moiety (D) is of Formula (IV-a),

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, D′ is D1.

In some embodiments, the PBD drug moiety (D) is of any one of formulae(V-1), (V-2), and (V-3):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the PBD drug moiety (D) is of Formula (VI-1):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the PBD drug moiety (D) is of Formula (VII),(VII-1), (VII-2), or (VII-3):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the PBD drug moiety (D) is of Formula (VIII):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, T is C₂₋₄ alkylene linker.

In some embodiments, A is

In some embodiments, A is

wherein each X₁ independently is CH or N.

In some embodiments, A is

wherein each X₁ independently is CH or N.

In some embodiments, A is:

wherein each X₁ independently is CH or N.

In some embodiments, E is —OH, —NH—(C₁₋₆ alkylene)-OH,

In some embodiments, E is

In some embodiments, the PBD drug moiety (D) is of any one of Formulae(IX-a) to (IX-r):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the PBD drug moiety (D), prior to being connectedto another portion of the conjugate, corresponds to a compound selectedfrom the compounds listed in Table 1, tautomers thereof,pharmaceutically acceptable salts or solvates thereof, orpharmaceutically acceptable salts or solvates of the tautomers.

In some embodiments, the PBD drug moiety (D), prior to being connectedto another portion of the conjugate, corresponds to a compound of anyone of Formula (XIIIa) to (XIIIm):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the PBD drug moiety (D) is selected from theconjugates listed in Table 1A, tautomers thereof, pharmaceuticallyacceptable salts or solvates thereof, and pharmaceutically acceptablesalts or solvates of the tautomers.

In some embodiments, the conjugate is selected from the conjugateslisted in Table 2, tautomers thereof, pharmaceutically acceptable saltsor solvates thereof, and pharmaceutically acceptable salts or solvatesof the tautomers.

In some aspects, the present disclosure provides a pharmaceuticalcomposition comprising the conjugate of any one of the preceding claimsand a pharmaceutically acceptable carrier.

In some aspects, the present disclosure provides a method of treating orpreventing a disease or disorder, comprising administering to a subjectin need thereof a pharmaceutically effective amount of the conjugate ofany one of the preceding claims.

In some embodiments, the disease or disorder is cancer.

In some aspects, the present disclosure provides a conjugate disclosedherein for use in treating or preventing a disease or disorder.

In some aspects, the present disclosure provides use of a conjugatedisclosed herein in treating or preventing a disease or disorder.

In some aspects, the present disclosure provides use of a conjugatedisclosed herein in the manufacture of a medicament for treating orpreventing a disease or disorder.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of tumor volume vs. time, showing the tumor response inmice inoculated subcutaneously with Calu-3 cells (n=10 for each group)after IV administration as a single dose on day 1 of vehicle ortrastuzumab-PBD conjugates: Example 4, Conjugate 14; Example 5,Conjugate 22; Example 6, Conjugate 28; and Example 7, Conjugate 34; eachat 1 mg/kg.

FIG. 2 is a plot of tumor volume vs. time, showing the tumor response inmice inoculated subcutaneously with Calu-3 cells (n=10 for each group)after IV administration as a single dose on day 1 of vehicle ortrastuzumab-PBD conjugates: Example 2, Conjugate 8, at 3 mg/kg and 6mg/kg; and Example 9, Conjugate 36, at 1 mg/kg and 3 mg/kg.

DETAILED DESCRIPTION

In some aspects, the present disclosure provides, inter alia, aconjugate (e.g., an antibody-drug conjugate (ADC)) of Formula (I):PBRM-[L^(C)-D]_(d15)  (I)or a pharmaceutically acceptable salt or solvate thereof, wherein:

PBRM denotes a protein based recognition-molecule;

L^(C) is a linker unit connecting the PBRM to D;

D is a PBD drug moiety; and

d₁₅ an integer from about 1 to about 20.

In some embodiments, the conjugates of Formula (I) include those whereeach of the moieties defined for one of PBRM, L^(C), D, and d₁₅ can becombined with any of the moieties defined for the others of PBRM, L^(C),D, and d₁₅.

In some embodiments, the PBRM is a targeting agent that binds to atarget moiety. In some embodiments, the PBRM is a cell binding agentspecifically binding to a cell component. In some embodiments, the PBRMspecifically binds to a target molecule of interest.

In some embodiments, the conjugate allows for delivery of the PBD drugmoiety (D) to a preferred site in a subject (e.g., a human). In someembodiments, the conjugate allows for the release of the PBD drug moiety(D) in an active form for its intended therapeutic effect.

In some embodiments, the conjugate comprises the PBD drug moiety (D)being covalently attached to a cell binding agent via the linker unit(L^(C)).

In some embodiments, the linker unit is a bifunctional ormultifunctional moiety which being capable of linking one or more PBDdrug moiety (D) and an antibody unit (Ab) to form an antibody-drugconjugate (ADC). The linker unit may be stable outside a cell (i.e.,extracellularly), or it may be cleavable by enzymatic activity,hydrolysis, or other metabolic conditions.

In some embodiments, the linker unit of the ADC prevents aggregation ofthe ADC and/or keep the ADC freely soluble in aqueous media and in amonomeric state.

In some embodiments, the linker unit of the ADC is stableextracellularly. In some embodiments, before transport or delivery intoa cell, the ADC is preferably stable and remains intact (i.e., theantibody remains linked to the drug moiety). In some embodiments, thelinker unit is stable outside the target cell and may be cleaved at anefficacious rate inside the cell. In some embodiments, the linker unitmay (i) maintain the specific binding properties of the antibody; (ii)allow for intracellular delivery of the conjugate or therapeutic agent;(iii) remain stable and intact (i.e., not cleaved) until the conjugatehas been delivered or transported to its targeted site; and/or (iv)maintain a cytotoxic, cell-killing effect or a cytostatic effect of thePBD drug moiety. Stability of the ADC may be measured by standardanalytical techniques such as mass spectroscopy, HPLC, and theseparation/analysis technique LC/MS.

Covalent attachment of the antibody and the PBD drug moiety requires thelinker unit to have two reactive functional groups (i.e., bivalency in areactive sense). Useful bivalent linker units for attaching two or morefunctional or biologically active moieties include, but are not limitedto, peptides, nucleic acids, drugs, toxins, antibodies, haptens, andreporter groups. Some known bivalent linker units and their resultingconjugates have been described (Hermanson, G. T. (1996) BioconjugateTechniques; Academic Press: New York, p 234-242).

In some embodiments, the linker unit may be substituted with one or moregroups which modulate aggregation, solubility, and/or reactivity. Insome embodiments, a sulfonate substituent may increase water solubilityof the reagent and facilitate the coupling reaction of the linkerreagent with the antibody or the PBD drug moiety, or facilitate thecoupling reaction of an antibody-linker reagent (Ab-L) with a PBD drugmoiety (D), or a PBD drug-linker reagent (D-L) with an antibody unit(Ab), depending on the synthetic route employed to prepare the ADC. Insome aspects, the present disclosure provides a method of preparing aconjugate (e.g., an antibody-drug conjugate (ADC)) of the presentdisclosure. Antibody-drug conjugates (ADC) can be conveniently preparedusing a linker unit having reactive functionality for binding to the PBDdrug moiety (D) and to the antibody unit (Ab). In some embodiments, acysteine thiol, or an amine (e.g. N-terminus or amino acid side chainsuch as lysine) of the antibody (Ab) can form a bond with a functionalgroup of a linker or spacer reagent, a PBD drug moiety (D), or a PBDdrug-linker reagent (D-RL).

Antibody-Drug Conjugate (ADC) Type I:

In some embodiments, the conjugate (e.g., the antibody-drug conjugate(ADC)) of the present disclosure is of Formula (II):

or a pharmaceutically acceptable salt or solvate thereof,wherein:

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM;

M^(P) is a Stretcher unit;

a₁ is an integer from 0 to 1;

M^(A) comprises a peptide moiety that contains at least two amino acids;

T′ is a hydrophilic group and the

between T′ and M^(A) denotes direct or indirect attachment of T′ andM^(A);

each occurrence of L^(D) is independently a divalent linker moietyconnecting D to M^(A) and comprises at least one cleavable bond suchthat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and

d₁₃ is an integer from 1 to 20.

In some embodiments, the conjugates of Formula (II) include those whereeach of the moieties defined for one of PBRM, D, L^(P′), L^(P), W^(P),M^(P), a₁, M^(A), T′, L^(D), and d₁₃ can be combined with any of themoieties defined for the others of PBRM, D, L^(P′), L^(P), W^(P), M^(P),a₁, M^(A), T′, L^(D), and d₁₃.

In some aspects, the present disclosure provides a scaffold of any oneof Formulae (IIa) to (IIe):

or a pharmaceutically acceptable salt or solvate thereof, wherein

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

L^(P′) is a divalent linker moiety connecting the PBRM to M; of whichthe corresponding monovalent moiety L^(P) contains a functional groupW^(P) that is capable of forming a covalent bond with a functional groupof the PBRM;

M^(P) is a Stretcher unit;

a₁ is an integer from 0 to 1; M^(A) comprises a peptide moiety thatcontains at least two amino acids;

T′ is a hydrophilic group and the

between T′ and M^(A) denotes direct or indirect attachment of T′ andM^(A);

each occurrence of W^(D) is independently a functional group that iscapable of forming a covalent bond with a functional group of D; eachoccurrence of L^(D) is independently a divalent linker moiety connectingW^(D) or D to M^(A) and L^(D) comprises at least one cleavable bond suchthat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and

d₁₃ is an integer from 1 to 10.

In some embodiments, the conjugates of any one of Formulae (IIa)-(IIe)include those where each of the moieties defined for one of PBRM, D,L^(P′), L^(P), W^(P), M^(P), a₁, M^(A), T′, L^(D), W^(D), and d₁₃ can becombined with any of the moieties defined for the others of PBRM, D,L^(P′), L^(P), W^(P), M^(P), a₁, M^(A), T′, L^(D), W^(D), and d₁₃.

The conjugates and scaffolds of the disclosure can include one or moreof the following features when applicable.

In some embodiments, d₁₃ is an integer from 2 to 14, from 2 to 12, from2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, from 4 to 10, from 4 to8, from 4 to 6, from 6 to 14, from 6 to 12, from 6 to 10, from 6 to 8,from 8 to 14, from 8 to 12, or from 8 to 10.

In some embodiments, di is an integer from 2 to 6 (e.g., d₁₃ is 2, 3, 4,5 or 6).

In some embodiments, d₁₃ is an integer from 2 to 4 (e.g., d₁₃ is 2, 3,or 4).

In some embodiments, d₁₃ is an integer from 4 to 6 (e.g., d₁₃ is 4, 5,or 6).

In some embodiments, d₁₃ is an integer from 6 to 8 (e.g., d₁₃ is 6, 7,or 8).

In some embodiments, di is an integer from 6 to 10 (e.g., d₁₃ is 6, 7,8, 9, or 10).

In some embodiments, do is 3 to 5.

In some embodiments, d₁₃ is 4 or 5.

L^(P) and L^(P′)

In some embodiments, L^(P′) is a divalent linker moiety connecting thePBRM to M^(P); of which the corresponding monovalent moiety is L^(P).

In some embodiments, L^(P), when not connected to PBRM, comprises aterminal group W^(P), in which each W^(P) independently is:

in which

R^(1K) is a leaving group (e.g., halide or RC(O)O— in which R ishydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety);

R^(1A) is a sulfur protecting group;

ring A is cycloalkyl or heterocycloalkyl;

ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R^(1J) is hydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety;

R^(2J) is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclicmoiety;

R^(4j) is C₁₋₆ alkyl; Z₁, Z₂, Z₃ and Z₇ are each independently a carbonor nitrogen atom; R⁴ is hydrogen, halogen, OR, —NO₂, —CN, —S(O)₂R, C₁₋₂₄alkyl (e.g., C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl, whereinthe C₁₋₂₄ alkyl (e.g., C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl,is optionally substituted with one or more aryl or heteroaryl; or twoR^(4j) together form an annelated cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, orheterocycloalkyl

R^(5j) is C(R^(4j))₂, O, S or NR; and

z₁ is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some embodiments, each R^(1K) is halo or RC(O)O— in which R ishydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety.

In some embodiments, each R^(1A) independently is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogenor an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkylmoiety.

In some embodiments, ring A is C₃₋₈ cycloalkyl or 5-19 memberedheterocycloalkyl.

In some embodiments, ring A is

wherein R^(6j) is hydrogen, halogen, C₁₋₂₄ alkyl (e.g., C₁₋₆ alkyl), or6-24 membered aryl or heteroaryl, wherein the C₁₋₂₄ alkyl (e.g., C₁₋₆alkyl), or 6-24 membered aryl or heteroaryl, is optionally substitutedwith one or more aryl or heteroaryl.

In some embodiments, ring A is

In some embodiments, ring A or B is C₃₋₈ cycloalkyl or 3-12 memberedheterocycloalkyl.

In some embodiments, ring A or B is piperazinyl or piperidinyl.

In some embodiments, each of R^(s1), R^(s2), and R^(s3) is hydrogen orC₁₋₆ alkyl.

In some embodiments, W^(P) is

In some embodiments, W^(P) is

In some embodiments, W^(P) is

L^(P′) comprises

In some embodiments, W^(P) is

In some embodiments, when W^(P) is

In some embodiments, when W^(P) is

In some embodiments, when W^(P) is

In some embodiments, when W^(P) is

L^(P′) comprise

In some embodiments, W^(P) is

wherein one of X_(a) and X_(b) is H and the other is a maleimidoblocking moiety. In some embodiments, a maleimido blocking compound(i.e., a compound that can react with maleimide to convert it tosuccinimide) may be used to quench the reaction between, e.g., theLinker-Drug moiety and PBRM, and a maleimido blocking moiety refers tothe chemical moiety attached to the succinimide upon conversion. In someembodiments, the maleimido blocking moieties are moieties that can becovalently attached to one of the two olefin carbon atoms upon reactionof the maleimido group with a thiol-containing compound of Formula(II′):R₉₀—(CH₂)_(d)—SH  (II′)wherein:

R₉₀ is NHR₉₁, OH, COOR₉₃, CH(NHR₉₁)COOR₉₃ or a substituted phenyl group;

R₉₃ is hydrogen or C₁₋₄ alkyl;

R₉₁ is hydrogen, CH₃ or CH₃CO and

d is an integer from 1 to 3.

In some embodiments, the maleimido blocking compound is cysteine,N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine,2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid,mercaptomethanol (i.e., HOCH₂SH), benzyl thiol in which phenyl issubstituted with one or more hydrophilic substituents, or3-aminopropane-1-thiol. The one or more hydrophilic substituents onphenyl comprise OH, SH, methoxy, ethoxy, COOH CHO, COC₁₋₄ alkyl, NH₂, F,cyano, SO₃H, PO₃H, and the like.

In some embodiments, the maleimido blocking group is —S—(CH₂)_(d)—R₉₀,wherein:

R₉₀ is OH, COOH, or CH(NHR₉₁)COOR₉₃;

R₉₃ is hydrogen or CH₃;

R₉₁ is hydrogen or CH₃CO; and

d is 1 or 2.

In some embodiments, the maleimido blocking group is —S—CH₂—CH(NH₂)COOH.

Stretcher Unit M^(P)

In some embodiments, M^(P), when present, is —(Z₄)—[(Z₅)—(Z₆)]_(z)—,with Z₄ connected to L^(P′) or L^(P) and Z₆ connected to M^(A); in which

z is 1, 2, or 3;

Z₄ is

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to Z₅ or Z₆ when present or to M^(A) when Z₅ and Z₆ are bothabsent;

-   -   b₁ is an integer from 0 to 6;    -   e₁ is an integer from 0 to 8,

R₁₇ is C₁₋₁₀ alkylene, C₁₋₁₀ heteroalkylene, C₃₋₈ cycloalkylene, O—(C₁₋₈alkylene, arylene, —C₁₋₁₀ alkylene-arylene-, -arylene-C₁₋₁₀ alkylene-,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-, —(C₃₋₈ cycloalkylene —C₁₋₁₀alkylene-, 4 to 14-membered heterocycloalkylene, —C₁₋₁₀ alkylene-(4 to14-membered heterocycloalkylene)-, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-, —C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀heteroalkylene-C(═O)—, —C₃₋₈ cycloalkylene-C(═O)—, —O—(C₁₋₈alkyl)-C(═O)—, -arylene-C(═O)—, —C₁₋₁₀ alkylene-arylene-C(═O)—, -arylene—C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-C(═O)—,—(C₃₋₈ cycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, -4 to 14-memberedheterocycloalkylene-C(═O)—, —C₁₋₁₀ alkylene-(4 to 14-memberedheterocycloalkylene)-C(═O)—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-NH—, —C₁₋₁₀heteroalkylene-NH—, —C₃₋₈ cycloalkylene-NH—, —O—(C₁₋₈ alkyl)-NH—,-arylene-NH—, —C₁₋₁₀ alkylene-arylene-NH—, -arylene-C₁₋₁₀ alkylene-NH—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-NH—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-NH—, -4 to 14-membered heterocycloalkylene-NH—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-NH—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-NH—, —C₁₋₁₀ alkylene-S—, —C₁₋₁₀heteroalkylene-S—, —C₃₋₈ cycloalkylene-S—, —O—C₁₋₈ alkyl)-S—,-arylene-S—, —C₁₋₁₀ alkylene-arylene-S—, -arylene-C₁₋₁₀ alkylene-S—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-S—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-S—, -4 to 14-membered heterocycloalkylene-S—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-S—, or -(4 to14-membered heterocycloalkylene)-C₁-C₁₀ alkylene-S—;

each Z₅ independently is absent, R₅₇—R₁₇ or a polyether unit:

each R₅₇ independently is a bond, NR₂₃, S or O;

each R₂₃ independently is hydrogen. C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl; and

each Z₆ independently is absent, —C₁₋₁₀ alkyl-R₃—, —C₁₋₁₀ alkyl-NR₅—,—C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-S— or —(C₁₋₁₀alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—;

each R₃ independently is —C(O)—NR₅— or —NR₅—C(O)—;

each R₅ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, COOH, or COO—C₁₋₆ alkyl; and

g₁ is an integer from 1 to 4.

In some embodiments, Z₄ is

e.g., wherein b₁ is 0, 1 or 4.

In some embodiments, Z₄ is

e.g., wherein b₁ is 1 or 4.

In some embodiments, Z₄ is

In some embodiments, Z₄ is

In some embodiments, each Z₅ independently is a polyalkylene glycol(PAO), including but are not limited to, polymers of lower alkyleneoxides, in particular polymers of ethylene oxide, such as, for example,propylene oxide, polypropylene glycols, polyethylene glycol (PEG),polyoxyethylenated polyols, copolymers thereof and block copolymersthereof. In some embodiments, the polyalkylene glycol is a polyethyleneglycol (PEG) including, but not limited to, polydisperse PEG,monodisperse PEG and discrete PEG. Polydisperse PEGs are a heterogeneousmixture of sizes and molecular weights whereas monodisperse PEGs aretypically purified from heterogeneous mixtures and are therefore providea single chain length and molecular weight. In some embodiments, the PEGunits are discrete PEGs provide a single molecule with defined andspecified chain length. In some embodiments, the polyethylene glycol ismPEG.

As used herein a subunit when referring to the PEG unit refers to apolyethylene glycol subunit having the formula

In some embodiments, the PEG unit comprises multiple PEG subunits.

In some embodiments, when z is 2 or 3, at least one Z₅ is a polyalkyleneglycol (PAO), e.g., a PEG unit.

In some embodiments, the PEG unit comprises 1 to 6 subunits.

In some embodiments, the PEG unit comprises 1 to 4 subunits.

In some embodiments, the PEG unit comprises 1 to 3 subunits.

In some embodiments, the PEG unit comprises 2 subunits.

In some embodiments, the PEG unit comprises 1 subunit.

In some embodiments, the PEG unit comprises one or multiple PEG subunitslinked together by a PEG linking unit. The PEG linking unit thatconnects one or more chains of repeating CH₂CH₂O— subunits can be Z₆. Insome embodiments, Z₆ is —C₁₋₁₀ alkyl-R₃—, —C₂₋₁₀ alkyl-NH—, —C₂₋₁₀alkyl-C(O)—, —C₂₋₁₀ alkyl-O— or —C₁₋₁₀ alkyl-S, wherein R₃ is —C(O)—NR₅—or —NR₅—C(O)—.

In some embodiments, the PEG linking unit is —C₁₋₁₀ alkyl-C(O)—NH— or—C₁₋₁₀ alkyl-NH—C(O)—. In one embodiment, the PEG linking unit is—(CH₂)₂—C(O)—NH—.

In some embodiments, each Z₅ is absent.

In some embodiments, when z is 2 or 3, at least one Z₅ is absent.

In some embodiments, each Z₅ is —(CH₂—CH₂—O—)₂—.

In some embodiments, when z is 2 or 3, at least one Z₅ is—(CH₂—CH₂—O—)₂—.

In some embodiments, each Z₅ independently is R₅₇—R₁₇. In someembodiments, each Z₅ independently is R₁₇, NHR₁₇, OR₁₇, or SR₁₇.

In some embodiments, when z is 2 or 3, at least one Z₅ is R₅₇—R₁₇, e.g.,R₁₇, NHR₁₇, OR₁₇, or SR₁₇.

In some embodiments, each Z₆ is absent.

In some embodiments, when z is 2 or 3, at least one Z₆ is absent.

In some embodiments, at least one of Z₅ and Z₆ is not absent.

In some embodiments, each Z (independently is —C₁₋₁₀ alkyl-R₃—, —C₁₋₁₀alkyl-NH—, —C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-S— or—(C₁₋₁₀ alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—. In some embodiments, g₁ is aninteger from 1 to 4.

In some embodiments, when z is 2 or 3, at least one Z₆ is —C₁₋₁₀alkyl-R₃—, —C₁₋₁₀ alkyl-NH—, —C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀alkyl-S— or —(C₁₋₁₀ alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—.

In some embodiments, g₁ is an integer from 1 to 4.

In some embodiments, each Z₆ independently or at least one Z₆ is —C₂₋₁₀alkyl-C(O)—, e.g., —(CH₂)₂—C(O)—.

In some embodiments, each Z₆ independently or at least one Z is —C₂₋₁₀alkyl-R₃—C₂₋₁₀ alkyl-C(O)—, e.g., —(CH₂)₂—C(O)NH—(CH₂)₂—C(O)—.

In some embodiments, each Z₆ independently or at least one Z₆ is —(C₂₋₁₀alkyl-R₃)_(g1)—C₂₋₁₀ alkyl-C(O)—, e.g.,—(CH₂)₂—C(O)NH—(CH₂)₂—NHC(O)—(CH₂)—C(O)—.

In some embodiments, —[(Z₅)—(Z₆)]_(z)— is not absent.

In some embodiments, —[(Z₅)—(Z₆)]_(z)— is a bond.

In some embodiments, —[(Z₅)—(Z₆)]_(z)— is—(CH₂CH₂O)₂—(CH₂)₂—C(O)—NH—(CH₂CH₂O)₂—.

In some embodiments, M^(P), when present, is

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M);

R₃, R₅, R₁₇, and R₂₃ are as defined herein;

R₄ is a bond or —NR₅—(CR₂₀R₂₁)—C(O)—;

each R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

each b₁ independently is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

each f₁ independently is an integer from 1 to 6; and

g₂ is an integer from 1 to 4.

In some embodiments, b₁ is 1.

In some embodiments, b₁ is 0

In some embodiments, each f₁ independently is 1 or 2.

In some embodiments, f₁ is 2.

In some embodiments, g₂ is 1 or 2.

In some embodiments, g₂ is 2.

In some embodiments, R₁₇ is unsubstituted.

In some embodiments, R₁₇ is optionally substituted.

In some embodiments, R₁₇ is optionally substituted by a basic unit,e.g., —(CH₂)_(x)NH₂, —(CH₂)_(x)NHR^(a), and —(CH₂)_(x)N(R^(a))₂, whereinx is an integer from 1 to 4 and each R^(a) is independently selectedfrom C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups are combinedwith the nitrogen to which they are attached to form an azetidinyl,pyrrolidinyl or piperidinyl group.

In some embodiments, R¹⁷ is —C₂₋₃ alkylene-C(═O)— wherein the alkyleneis optionally substituted by a basic unit, e.g., —(CH₂)_(x)NH₂,—(CH₂)_(x)NHR^(a), and —(CH₂)_(x)N(R^(a))₂, wherein x and R^(a) are asdefined herein.

In some embodiments, wherein M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to M^(A).

In some embodiments, wherein M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to M^(A).

In some embodiments, wherein M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to M^(A).M^(A)

In some embodiments, M^(A) is a linker moiety that is capable ofconnecting one or more drugs and one or more hydrophilic groups to L^(P)or L^(P′) In some embodiments, M^(A) comprises a peptide moiety of atleast two amino acid (AA) units.

The peptide moiety is a moiety that is capable of forming a covalentbond with a -L^(D)-D unit and allows for the attachment of multipledrugs. In some embodiments, peptide moiety comprises a single AA unit orhas two or more AA units (e.g., 2 to 10, preferably from 2 to 6, e.g.,2, 3, 4, 5 or 6) wherein the AA units are each independently a naturalor non-natural amino acid, an amino alcohol, an amino aldehyde, adiamine, or a polyamine or combinations thereof. If necessary in orderto have the requisite number of attachments, at least one of AA unitswill have a functionalized side chain to provide for attachment of the-L^(D)-D unit. Exemplary functionalized AA units (e.g., amino acids,amino alcohols, or amino aldehydes) include, for example, azido oralkyne functionalized AA units (e.g., amino acid, amino alcohol, oramino aldehyde modified to have an azide group or alkyne group forattachment using click chemistry).

In some embodiments, the peptide moiety has 2 to 12 AA units.

In some embodiments, the peptide moiety has 2 to 10 AA units.

In some embodiments, the peptide moiety has 2 to 6 AA units.

In some embodiments, the peptide moiety has 2, 3, 4, 5 or 6 AA units.

In some embodiments, an AA unit has three attachment sites, (e.g., forattachment to L^(M), the hydrophilic group (T′) or another AA unit, andto the -L^(D)-D unit). In some embodiments, the AA unit has the formula:

wherein the wavy line indicates attachment sites within the conjugate(e.g., the antibody-drug conjugate (ADC)) of the disclosure orintermediates thereof; and R₁₀₀ and R₁₁₀ are as defined herein.

In some embodiments, an AA unit has two attachment sites (i.e., aterminal unit) and one of the attachment sites shown above can replaced,for example, by H, OH, or an unsubstituted C₁₋₃ alkyl group.

In some embodiments, the peptide moiety comprises at least two AA unitsof the following formula:

wherein:

each R₁₁₁ independently is H, p-hydroxybenzyl, methyl, isopropyl,isobutyl, sec-butyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂,—CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl,

the wavy line indicates the attachment sites within the conjugate orintermediates thereof; and

R₁₀₀ and R₁₁₀ are as defined herein.

In some embodiments, the peptide moiety comprises at least two AA units,e.g., cysteine-alanine is:

wherein the wavy lines and asterisk indicates attachment sites withinthe conjugate or intermediates thereof. For example, asterisk indicatesattachment site of -L^(D)-D unit or a hydrophilic group. For example,the wavy line next to the carbonyl group indicates attachment site of-L^(D)-D unit or a hydrophilic group. For example, the wavy line next tothe amine group indicates attachment site of -L^(D)-D unit or ahydrophilic group. For example, one or two of the wavy lines andasterisk indicate attachment site(s) of one or more -L^(D)-D units orone or more hydrophilic groups.

In some embodiments, the peptide moiety comprises at least two AA units,which provide two attachment sites, e.g., cysteine-alanine is:

wherein the wavy line and asterisk indicates attachment sites within theconjugate or intermediates thereof. In some embodiments, asteriskindicates attachment site of -L^(D)-D unit or a hydrophilic group. Insome embodiments, the wavy line indicates attachment site of -L^(D)-Dunit or a hydrophilic group.

One or more AA units (e.g., an amino acid, amino alcohol, amino aldehydeor polyamine) of the peptide moiety can be replaced by an optionallysubstituted C₁₋₂₀ heteroalkylene (e.g., optionally substituted C₁₋₁₂heteroalkylene), optionally substituted C₃₋₈ heterocyclo, optionallysubstituted C₆₋₁₄ arylene, or optionally substituted C₃₋₈ carbocyclo asdescribed herein. The optionally substituted heteroalkylene,heterocycle, arylene or carbocyclo may have one or more functionalgroups for attachment within a conjugate or intermediates thereof.Suitable substituents include, but are not limited to (═O), —R^(1C),—R^(1B), —OR^(1B), —SR^(1B), —N(R^(1B))₂, —N(R^(1B))₃, ═NR^(1B),C(R^(1C))₃, CN, OCN, SCN, N═C═O, NCS, NO, NO₂, ═N₂, N₃,NR^(1B)C(═O)R^(1B), —C(═O)R^(1B), —C(═O)N(R^(1B))₂, SO₃ ⁻, SO₃H,S(═O)₂R^(1B), —OS(═O)₂OR^(1B), —S(═O)₂NR^(1B), —S(═O)R^(1B),—OP(═O)(OR^(1B)), —P(═O)(OR^(1B))₂, PO₃ ⁻, PO₃H₂, AsO₂H₂, C(═O)R^(1B),C(═O)R^(1C), C(═S)R^(1B), CO₂R^(1B), CO₂—, C(═S)OR^(1B), C(═O)SR^(1B),C(═S)SR^(1B), C(═O)N(R^(1B))₂, C(═S)N(R^(1B))₂, andC(═NR^(1B))N(R^(1B))₂, wherein each R^(1C) is independently a halogen(e.g., —F, —CI, —Br, or —I), and each R^(1B) is independently —H, —C₁₋₂₀alkyl, —C₆₋₂₀ aryl, —C₃₋₁₄ heterocycle, a protecting group or a prodrugmoiety.

In some embodiments, the one or more substituents for theheteroalkylene, heterocycle, arylene or carbocyclo are selected from(═O), R^(1C), R^(1B), OR^(1B), SR^(1B), and N(R^(1B))₂.

In some embodiments, the peptide moiety can be a straight chain orbranched moiety of having the Formula:

wherein:

each BB′ is independently an amino acid, optionally substituted C₁₋₂₀heteroalkylene (e.g., optionally substituted C₁₋₁₂ heteroalkylene),optionally substituted C₃₋₈ heterocyclo, optionally substituted C₆₋₁₄arylene, or optionally substituted C₃-C₈ carbocyclo;

d₁₂ is an integer from 1 to 10; and

the wavy line indicates the covalent attachment sites within theconjugate or intermediate thereof.

In some embodiments, d₁₂ is an integer from 2 to 10.

In some embodiments, d₁₂ is an integer from 2 to 6.

In some embodiments, d₁₂ is an integer from 4, 5 or 6.

In some embodiments, di is an integer from 5 or 6.

In some embodiments, the optionally substituted heteroalkylene,heterocycle, arylene or carbocyclo have functional groups forattachments between the BB′ subunits and/or for attachments within aconjugate or intermediates thereof disclosed herein.

In some embodiments, the peptide moiety comprises no more than 2optionally substituted C₁₋₂₀ heteroalkylenes, optionally substitutedC₃₋₁₈ heterocyclos, optionally substituted C₆₋₁₄ arlenes, or optionallysubstituted C₃₋₈ carbocyclos.

In other embodiments, the peptide moiety comprises no more than 1optionally substituted C₁₋₂₀ heteroalkylenes, optionally substitutedC₃₋₈ heterocyclos, optionally substituted C₆₋₁₄ arylenes, or optionallysubstituted C₃₋₈ carbocyclos. The optionally substituted heteroalkylene,heterocycle, arylene or carbocyclo will have functional groups forattachment between the BB′ subunits and/or for attachments within aconjugate or intermediates thereof disclosed herein.

In some embodiments, at least one BB′ is an amino acid. In someembodiments, the amino acid can be an alpha, beta, or gamma amino acid,which can be natural or non-natural. The amino acid can be a D or Lisomer.

In some embodiments, attachment within the peptide moiety or with theother components of the conjugate (or intermediate thereof, or scaffold)can be, for example, via amino, carboxy, or other functionalities.

In some embodiments, each amino acid of the peptide moiety can beindependently D or L isomer of a thiol containing amino acid. The thiolcontaining amino acid can be, for example, cysteine, homocysteine, orpenicillamine.

In some embodiments, each amino acid that comprises the peptide moietycan be independently the L- or D-isomers of the following amino acids:alanine (including β-alanine), arginine, aspartic acid, asparagine,cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine,lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan,proline, ornithine, penicillamine, aminoalkynoic acid, aminoalkanedioicacid, heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoicacid, stereoisomers thereof (e.g., isoaspartic acid and isoglutamicacid), and derivatives thereof.

In some embodiments, each amino acid that comprises the peptide moietyis independently cysteine, homocysteine, penicillamine, ornithine,lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid,glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine,methionine, valine, alanine, or a stereoisomers thereof (e.g.,isoaspartic acid and isoglutamic acid).

In some embodiments, the peptide moiety comprises a monopeptide, adipeptide, tripeptide, tetrapeptide, or pentapeptide.

In some embodiments, the peptide moiety contains at least about fiveamino acids (e.g., 5, 6, 7, 8, 9, or 10 amino acids).

In some embodiments, the peptide moiety contains at most about ten aminoacids.

In some embodiments, the peptide moiety comprises a pentapeptide.

In some embodiments, each amino acid that comprises the peptide moietyis independently glycine, serine, glutamic acid, lysine, aspartic acidand cysteine.

In some embodiments, the peptide moiety comprises at least four glycinesand at least one serine, e.g., (glycine)₄ and serine wherein the serineis at any position along the peptide chain, such as, for example,(serine)-(glycine)₄; (glycine)-(serine)-(glycine)₃;(glycine)₂-(serine)-(glycine)₂; (glycine)₃-(serine)-(glycine); or(glycine)₄-(serine).

In some embodiments, the peptide moiety comprises (glycine)₄-(serine) or(serine)-(glycine)₄.

In some embodiments, the peptide moiety comprises at least four glycinesand at least one glutamic acid e.g., (glycine)₄ and glutamic acidwherein the glutamic acid is at any position along the peptide chain,such as, for example, (glutamic acid)-(glycine; (glycine)-(glutamicacid)-(glycine)₃; (glycine)₂-(glutamic acid)-(glycine)₂;(glycine)₃-(glutamic acid)-(glycine); or (glycine)₄-(glutamic acid).

In some embodiments, the peptide moiety comprises (glutamicacid)-(glycine)₄; or (glycine)₄-(glutamic acid).

In some embodiments, the peptide moiety comprises(β-alanine)-(glycine)₄-(serine) wherein the serine is at any positionalong the peptide chain, such as, for example,(β-alanine)-(serine)-(glycine)₄;(β-alanine)-(glycine)-(serine)-(glycine)₃;(β-alanine)-(glycine)₂-(serine)-(glycine)₂;(β-alanine)-(glycine)₃-(serine)-(glycine); or(β-alanine)-(glycine)₄-(serine).

In some embodiments, the peptide moiety comprises(glycine)₄-(serine)-(glutamic acid) wherein the serine is at anyposition along the peptide chain, such as, for example,(serine)-(glycine)₄-(glutamic acid);(glycine)-(serine)-(glycine)₃-(glutamic acid);(glycine)₂-(serine)-(glycine)₂-(glutamic acid);(glycine)₃-(serine)-(glycine)-(glutamic acid); or(glycine)₄-(serine)-(glutamic acid). In another embodiment, the peptidemoiety comprises (β-alanine)-(glycine)₄-(serine)-(glutamic acid) whereinthe serine is at any position along the peptide chain, such as, forexample, (β-alanine)-(serine)-(glycine)₄-(glutamic acid);(β-alanine)-(glycine)-(serine)-(glycine)₃-(glutamic acid);(β-alanine)-(glycine)₂-(serine)-(glycine)₂-(glutamic acid);(β-alanine)-(glycine)₃-(serine)-(glycine)-(glutamic acid); or(β-alanine)-(glycine)₄-(serine)-(glutamic acid).

In some embodiments, when at least one of hydrophilic groups (T′) is apolyalcohol or derivative thereof (e.g., an amino polyalcohol) or aglucosyl-amine or a di-glucosyl-amine or a tri-glucosyl-amine, M^(A)does not have to comprise a peptide moiety. In some embodiments, M^(A)comprises one or more of the following:

wherein

the wavy line indicates attachment sites within the conjugate (e.g., theantibody-drug conjugate (ADC)) of the disclosure or intermediatesthereof; R₁₀₀ and R₁₁₀ and R₁₀ are as defined herein.

In some embodiments, R₁₁₀ is:

wherein the asterisk indicates attachment to the carbon labeled x andthe wavy line indicates one of the three attachment sites.

In some embodiments, R₁₀₀ is independently selected from hydrogen andCH₃.

In some embodiments, Y is N.

In some embodiments, Y is CH.

In some embodiments, R₁₀₀ is H or CH₃.

In some embodiments, each c′ is independently an integer from 1 to 3.

In some embodiments, R₁₁₀ is not

L^(D) and W^(D)

In some embodiments, each occurrence of L^(D) is independently adivalent linker moiety connecting D to M^(A) and comprises at least onecleavable bond such that when the bond is broken, D is released in anactive form for its intended therapeutic effect.

In some embodiments, L^(D) is a component of the Releasable AssemblyUnit. In other embodiments, L^(D) is the Releasable Assembly Unit.

In some embodiments, L^(D) comprises one cleavable bond.

In some embodiments, L^(D) comprises multiple cleavage sites or bonds.

Functional groups for forming a cleavable bond can include, for example,sulfhydryl groups to form disulfide bonds, aldehyde, ketone, orhydrazine groups to form hydrazone bonds, hydroxylamine groups to formoxime bonds, carboxylic or amino groups to form peptide bonds,carboxylic or hydroxy groups to form ester bonds, and sugars to formglycosidic bonds. In some embodiments, L^(D) comprises a disulfide bondthat is cleavable through disulfide exchange, an acid-labile bond thatis cleavable at acidic pH, and/or bonds that are cleavable by hydrolases(e.g., peptidases, esterases, and glucuronidases). In some embodiments,L^(D) comprises a carbamate bond (i.e., —O—C(O)—NR—, in which R is H oralkyl or the like).

The structure and sequence of the cleavable bond(s) in L^(D) can be suchthat the bond(s) is cleaved by the action of enzymes present at thetarget site. In other embodiments, the cleavable bond(s) can becleavable by other mechanisms.

In some embodiments, the cleavable bond(s) can be enzymatically cleavedby one or more enzymes, including a tumor-associated protease, toliberate the Drug moiety or D, which in one embodiment is protonated invivo upon release to provide a Drug moiety or D.

In certain embodiments, L^(D) can comprise one or more amino acids. Insome embodiments, each amino acid in L^(D) can be natural or unnaturaland/or a D- or L-isomer provided that there is a cleavable bond. In someembodiments, L^(D) comprising an alpha, beta, or gamma amino acid thatcan be natural or non-natural. In some embodiments, L^(D) comprises 1 to12 (e.g., 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12) amino acids in contiguous sequence. In certainembodiments, L^(D) can comprise only natural amino acids. In otherembodiments, L^(D) can comprise only non-natural amino acids. In someembodiments, L^(D) can comprise a natural amino acid linked to anon-natural amino acid. In some embodiments, L^(D) can comprise anatural amino acid linked to a D-isomer of a natural amino acid. Anexemplary L^(D) comprises a dipeptide such as -Val-Cit-, -Phe-Lys-,-Ala-Ala- or -Val-Ala-.

In some embodiments, L^(D) comprises, a monopeptide, a dipeptide, atripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, aheptapeptide, an octapeptide, a nonapeptide, a decapeptide, anundecapeptide or a dodecapeptide unit.

In some embodiments, L^(D) comprises a peptide (e.g., of 1 to 12 aminoacids), which is conjugated directly to the drug moiety. In some suchembodiments, the peptide is a single amino acid or a dipeptide.

In some embodiments, each amino acid in L^(D) is independently selectedfrom alanine, -alanine, arginine, aspartic acid, asparagine, histidine,glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine,serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine,cysteine, methionine, selenocysteine, ornithine, penicillamine,aminoalkanoic acid, aminoalkynoic acid, aminoalkanedioic acid,aminobenzoic acid, amino-heterocyclo-alkanoic acid,heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoic acid,and derivatives thereof.

In some embodiments, each amino acid is independently selected fromalanine, β-alanine, arginine, aspartic acid, asparagine, histidine,glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine,serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine,cysteine, methionine, citrulline and selenocysteine.

In some embodiments, each amino acid is independently selected from thegroup consisting of alanine, β-alanine, arginine, aspartic acid,asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine,lysine, leucine, serine, tyrosine, threonine, isoleucine, proline,tryptophan, valine, citrulline and derivatives thereof.

In some embodiments, each amino acid is selected from the proteinogenicor the non-proteinogenic amino acids.

In some embodiments, each amino acid in L^(D) can be independentlyselected from L- or D-isomers of the following amino acids: alanine,β-alanine, arginine, aspartic acid, asparagine, cysteine, histidine,glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine,methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine,penicillamine, aminoalkynoic acid, aminoalkanedioic acid,heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoic acid,valine, citrulline or derivatives thereof.

In some embodiments, each amino acid in L^(D) is independently cysteine,homocysteine, penicillamine, ornithine, lysine, serine, threonine,glycine, glutamine, alanine, aspartic acid, glutamic acid,selenocysteine, proline, glycine, isoleucine, leucine, methionine,valine, citrulline or alanine.

In some embodiments, each amino acid in L^(D) is independently selectedfrom L-isomers of the following amino acids: alanine, β-alanine,arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid,glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine,isoleucine, tryptophan, citrulline or valine.

In some embodiments, each amino acid in L^(D) is independently selectedfrom D-isomers of the following amino acids: alanine, β-alanine,arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid,glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine,isoleucine, tryptophan, citrulline or valine.

In some embodiments, each amino acid in L^(D) is alanine, β-alanine,glycine, glutamic acid, isoglutamic acid, isoaspartic acid, valine,citrulline or aspartic acid.

In one embodiment, L^(D) comprises β-alanine.

In another embodiment, L^(D) comprises (β-alanine)-(alanine).

In another embodiment, L^(D) comprises (β-alanine)-(glutamic acid).

In another embodiment, L^(D) comprises (β-alanine)-(isoglutamic acid).

In another embodiment, L^(D) comprises (β-alanine)-(aspartic acid).

In another embodiment, L^(D) comprises (β-alanine)-(isoaspartic acid).

In another embodiment, L^(D) comprises (β-alanine)-(valine).

In another embodiment, L^(D) comprises (β-alanine)-(valine)-(alanine).

In another embodiment, L^(D) comprises (β-alanine)-(alanine)-(alanine).

In another embodiment, L^(D) comprises (β-alanine)-(valine)-(citruline).

In another embodiment, L^(D) comprises (β-alanine)-(valine)-(lys).

In another embodiment, L^(D) comprises (β-alanine)-(lys).

In another embodiment, L^(D) comprises (β-alanine)-(gly)-(gly)-(gly).

In some embodiments, L^(D) comprises:

-   -   (i) (β-alanine)-(alanine)-(alanine); or    -   (ii) (β-alanine)-(valine)-(alanine).

In some embodiments, L^(D) comprises a carbamate bond in addition to oneor more amino acids.

In some embodiments, L^(D) can be designed and optimized in theirselectivity for enzymatic cleavage by a particular enzyme, e.g., atumor-associated protease.

In some embodiments, L^(D) comprises a bond whose cleavage is catalyzedby cathepsin B, C and D, or a plasmin protease.

In some embodiments, L^(D) comprises a sugar cleavage site. In some suchembodiments, L^(D) comprises a sugar moiety (Su) linked via an oxygenglycosidic bond to a self-immolative group. A “self-immolative group”can be a tri-functional chemical moiety that is capable of covalentlylinking together three spaced chemical moieties (i.e., the sugar moiety(via a glycosidic bond), a drug moiety (directly or indirectly), andM^(A) (directly or indirectly). The glycosidic bond will be one that canbe cleaved at the target site to initiate a self-immolative reactionsequence that leads to a release of the drug.

In some embodiments, L^(D) comprises a sugar moiety (Su) linked via aglycoside bond (—O′—) to a self-immolative group (K) of the formula:

wherein the self-immolative group (K) forms a covalent bond with thedrug moiety (directly or indirectly) and also forms a covalent bond withM^(A) (directly or indirectly). Examples of self-immolative groups aredescribed in, e.g., WO 2015/057699, the contents of which are herebyincorporated by reference in its entirety.

In some embodiments, when not connected to or prior to connecting to thePBD drug moiety, L^(D) comprises a functional a functional group W^(D).Each W^(D) independently can be a functional group as listed for W^(P).In some embodiments, each W independently is

in which R^(1A) is a sulfur protecting group, each of ring A and B,independently, is cycloalkyl or heterocycloalkyl, R^(W) is an aliphatic,heteroaliphatic, carbocyclic or heterocycloalkyl moiety; ring D isheterocycloalkyl; R^(1J) is hydrogen or an aliphatic, heteroaliphatic,carbocyclic, or heterocycloalkyl moiety; and R^(1K) is a leaving group(e.g., halide or RC(O)— in which R is hydrogen or an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety).

In some embodiments, W^(D) is

In some embodiments, W^(D) is

wherein one of X_(a) and X_(b) is H and the other is a maleimidoblocking moiety.

In some embodiments, W^(D) is

T′

In some embodiments, the hydrophilic group (T′) included in theconjugates or scaffolds of the disclosure is a water-soluble andsubstantially non-antigenic polymer. Examples of the hydrophilic group,include, but are not limited to, polyalcohols, polyethers, polyanions,polycations, polyphosphoric acids, polyamines, polysaccharides,polyhydroxy compounds, polylysines, and derivatives thereof. One end ofthe hydrophilic group (T′) can be functionalized so that it can becovalently attached to the Multifunctional Linker or M^(A) linker (e.g.,to an amino acid in the M^(A) linker) by means of a non-cleavablelinkage or via a cleavable linkage. Functionalization can be, forexample, via an amine, thiol, NHS ester, maleimide, alkyne, azide,carbonyl, or other functional group. The other terminus (or termini) ofthe hydrophilic group (T′) will be free and untethered. By “untethered”,it is meant that the hydrophilic group (T′) will not be attached toanother moiety, such as D or a Drug Moiety, Releasable Assembly Unit, orother components of the conjugates or scaffolds of the disclosure. Thefree and untethered end of the hydrophilic group (T′) may include amethoxy, carboxylic acid, alcohol or other suitable functional group.The methoxy, carboxylic acid, alcohol or other suitable functional groupacts as a cap for the terminus or termini of the hydrophilic group.

A cleavable linkage refers to a linkage that is not substantiallysensitive to cleavage while circulating in the plasma but is sensitiveto cleavage in an intracellular or intratumoral environment. Anon-cleavable linkage is one that is not substantially sensitive tocleavage in any biological environment. Chemical hydrolysis of ahydrazone, reduction of a disulfide, and enzymatic cleavage of a peptidebond or glycosidic linkage are examples of cleavable linkages. Exemplaryattachments of the hydrophilic group (T′) are via amide linkages, etherlinkages, ester linkages, hydrazone linkages, oxime linkages, disulfidelinkages, peptide linkages or triazole linkages. In some embodiments,the attachment of the hydrophilic group (T′) to the MultifunctionalLinker or M^(A) linker (e.g., to an amino acid in the M^(A) linker) isvia an amide linkage.

For those embodiments wherein the conjugate or scaffold of thedisclosure comprises more than one hydrophilic groups, the multiplehydrophilic groups may be the same or different chemical moieties (e.g.,hydrophilic groups of different molecular weight, number of subunits, orchemical structure). The multiple hydrophilic groups can be attached tothe Multifunctional Linker or M^(A) linker at a single attachment siteor different sites.

The addition of the hydrophilic group (T′) may have two potentialimpacts upon the pharmacokinetics of the resulting conjugate. Thedesired impact is the decrease in clearance (and consequent in increasein exposure) that arises from the reduction in non-specific interactionsinduced by the exposed hydrophobic elements of the drug or drug-linker.The second impact is undesired impact and is the decrease in volume andrate of distribution that may arise from the increase in the molecularweight of the conjugate. Increasing the molecular weight of thehydrophilic group (T′) increases the hydrodynamic radius of a conjugate,resulting in decreased diffusivity that may diminish the ability of theconjugate to penetrate into a tumor. Because of these two competingpharmacokinetic effects, it is desirable to use a hydrophilic group (T′)that is sufficiently large to decrease the conjugate clearance thusincreasing plasma exposure, but not so large as to greatly diminish itsdiffusivity, which may reduce the ability of the conjugate to reach theintended target cell population.

In some embodiments, the hydrophilic group, includes, but is not limitedto, a sugar alcohol (also known as polyalcohol, polyhydric alcohol,alditol or glycitol, such as inositol, glycerol, erythritol, threitol,arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, and thelike) or a derivative thereof (e.g., amino polyalcohol), carbohydrate(e.g., a saccharide), a polyvinyl alcohol, a carbohydrate-based polymer(e.g., dextrans), a hydroxypropylmethacrylamide (HPMA), a polyalkyleneoxide, and/or a copolymer thereof.

In some embodiments, the hydrophilic group (T′) comprises a plurality ofhydroxyl (“—OH”) groups, such as moieties that incorporatemonosaccharides, oligosaccharides, polysaccharides, and the like. In yetanother embodiment the hydrophilic group (T′) comprises a plurality of—(CR₅₈OH)— groups, wherein R₅₈ is hydrogen or C₁₋₈ alkyl.

In some embodiments, the hydrophilic group (T′) comprises one or more ofthe following fragments of the formula:

in which

n₁ is an integer from 0 to about 6;

each R₅₈ is independently hydrogen or C₁₋₈ alkyl;

R₆₀ is a bond, a C₁₋₆ alkyl linker, or —CHR₅₉— in which R₅₉ is H, alkyl,cycloalkyl, or arylalkyl;

R₆₁ is CH₂OR₆₂, COOR₆₂, —(CH₂)_(n2)COOR₆₂, or a heterocycloalkylsubstituted with one or more hydroxyl;

R₆₂ is H or C₁₋₈ alkyl; and

n₂ is an integer from 1 to about 5.

In some embodiments, R₅₈ is hydrogen, R₆₀ is a bond or a C₁₋₆ alkyllinker, n₁ is an integer from 1 to about 6, and R₆₁ is CH₂OH or COOH. Insome embodiments, R₅₈ is hydrogen, R₆₀ is —CHR₅₉—, n₁ is 0, and R₆₁ is aheterocycloalkyl substituted with one or more hydroxyl, e.g., amonosaccharide.

In some embodiments, the hydrophilic group (T′) comprises aglucosyl-amine, a diamine or a tri-amine.

In some embodiments, the hydrophilic group (T′) comprises one or more ofthe following fragments or a stereoisomer thereof:

wherein:

R₅₉ is H, alkyl, cycloalkyl, or arylalkyl;

n₁ is an integer from 1 to about 6;

n₂ is an integer from 1 to about 5; and

n₃ is an integer from about 1 to about 3.

It is understood that all stereochemical forms of the hydrophilic groupsare contemplated herein. In some embodiments, in the above formula, thehydrophilic group (T′) may be derived from ribose, xylose, glucose,mannose, galactose, or other sugar and retain the stereochemicalarrangements of pendant hydroxyl and alkyl groups present on thosemolecules. In addition, it is to be understood that in the foregoingformulae, various deoxy compounds are also contemplated. Illustratively,one or more of the following features are contemplated for thehydrophilic groups when applicable:

In some embodiments, n₃ is 2 or 3.

In some embodiments, n₁ is 1, 2, or 3.

In some embodiments, n₂ is 1.

In some embodiments, R₅₉ is hydrogen.

In some embodiments, the hydrophilic group (T′) comprises:

In some embodiments, the hydrophilic group (T′) comprises:

In some embodiments, the hydrophilic group (T′) comprises:

In some embodiments, the hydrophilic group (T′) comprises

in which

n₄ is an integer from 1 to about 25;

each R₆₃ is independently hydrogen or C₁₋₈ alkyl;

R₆₄ is a bond or a C₁₋₈ alkyl linker;

R₆₅ is H, C₁₋₈ alkyl, —(CH₂)_(n2)COOR₆₂, or —(CH₂)_(n2)COR₆₆;

R₆₂ is H or C₁₋₈ alkyl;

R₆₆ is

and

n₂ is an integer from 1 to about 5.

In some embodiments, the hydrophilic group (T′) comprises:

In some embodiments, n₄ is an integer from about 2 to about 20, fromabout 4 to about 16, from about 6 to about 12, from about 8 to about 12.

In some embodiments, n₄ is 6, 7, 8, 9, 10, 11, or 12.

In some embodiments, n₄ is 8 or 12.

In some embodiments, the hydrophilic group (T′) comprises:

in which n₄ is an integer from about 2 to about 20, from about 4 toabout 16, from about 6 to about 12, from about 8 to about 12.

In some embodiments, n₄ is 6, 7, 8, 9, 10, 11, or 12.

In some embodiments, n₄ is 8 or 1

In some embodiments, the hydrophilic group (T′) comprises a polyether,e.g., a polyalkylene glycol (PAO). PAO includes but is not limited to,polymers of lower alkylene oxides, in particular polymers of ethyleneoxide, such as, for example, propylene oxide, polypropylene glycols,polyethylene glycol (PEG), polyoxyethylenated polyols, copolymersthereof and block copolymers thereof. In other embodiments thepolyalkylene glycol is a polyethylene glycol (PEG) including, but notlimited to, polydisperse PEG, monodisperse PEG and discrete PEG.Polydisperse PEGs are a heterogeneous mixture of sizes and molecularweights whereas monodisperse PEGs are typically purified fromheterogeneous mixtures and are therefore provide a single chain lengthand molecular weight. In another embodiment, the PEG units are discretePEGs provide a single molecule with defined and specified chain length.In some embodiments, the polyethylene glycol is mPEG.

In some embodiments, the hydrophilic group (T′) comprises a PEG unitwhich comprises one or multiple polyethylene glycol chains. Thepolyethylene glycol chains can be linked together, for example, in alinear, branched or star shaped configuration. The PEG unit, in additionto comprising repeating polyethylene glycol subunits, may also containnon-PEG material (e.g., to facilitate coupling of multiple PEG chains toeach other or to facilitate coupling to the amino acid). Non-PEGmaterial refers to the atoms in the PEG chain that are not part of therepeating —CH₂CH₂O— subunits. In one embodiment, the PEG chain cancomprise two monomeric PEG chains linked to each other via non-PEGelements. In another embodiment, the PEG Unit can comprise two linearPEG chains attached to a central core that is attached to the amino acid(i.e., the PEG unit itself is branched).

The PEG unit may be covalently bound to the Multifunctional Linker orM^(A) linker (e.g., to an amino acid in the M^(A) linker) via a reactivegroup. Reactive groups are those to which an activated PEG molecule maybe bound (e.g., a free amino or carboxyl group). For example, N-terminalamino acids and lysines (K) have a free amino group; and C-terminalamino acid residues have a free carboxyl group. Sulfhydryl groups (e.g.,as found on cysteine residues) may also be used as a reactive group forattaching PEG.

In some embodiments, the PEG unit may be attached to the MultifunctionalLinker or M^(A) linker (e.g., to an amino acid in the M^(A) linker) byusing methoxylated PEG (“mPEG”) having different reactive moieties,including, but not limited to, succinimidyl succinate (SS), succinimidylcarbonate (SC), mPEG-imidate, para-nitrophenylcarbonate (NPC),succinimidyl propionate (SPA), and cyanuric chloride. Examples of mPEGsinclude, but are not limited to, mPEG-succinimidyl succinate (mPEG-SS),mPEG₂-succinimidyl succinate (mPEG₂-SS), mPEG-succinimidyl carbonate(mPEG-SC), mPEG₂-succinimidyl carbonate (mPEG₂-SC), mPEG-imidate,mPEG-para-nitrophenylcarbonate (mPEG-NPC), mPEG-imidate,mPEG₂-para-nitrophenylcarbonate (mPEG₂-NPC), mPEG-succinimidylpropionate (mPEG-SPA), mPEG₂-succinimidyl propionate (mPEG₂-SPA),mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG₂-N-hydroxy-succinimide(mPEG-NHS), mPEG-cyanuric chloride, mPEG₂-cyanuric chloride,mPEG₂-Lysinol-NPC, and mPEG₂-Lys-NHS. A wide variety of PEG species canbe used, and substantially any suitable reactive PEG reagent can beused. In some embodiments, the reactive PEG reagent will result information of a carbamate or amide bond upon attachment to theMultifunctional Linker or M^(A) linker (e.g., to an amino acid in theM^(A) linker). The reactive PEG reagents include, but are not limitedto, mPEG₂-N-hydroxy-succinimide (mPEG₂-NHS), bifunctional PEGpropionaldehyde (mPEG₂-ALD), multi-Arm PEG, maleimide-containing PEG(mPEG(MAL)₂, mPEG₂(MAL)), mPEG-NH₂, mPEG-succinimidyl propionate(mPEG-SPA), succinimide of mPEG butanoate acid (mPEG-SBA),mPEG-thioesters, mPEG-Double Esters, mPEG-BTC, mPEG-ButyrALD,mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEGs(e.g., NH₂-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinylsulfone(NHS-PEG-VS), or NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS),PEG-phospholipids (e.g., mPEG-DSPE), multi-armed PEGs of the SUNBRITE™series including the glycerine-based PEGs activated by a chemistrychosen by those skilled in the art, any SUNBRITE activated PEGs(including but not limited to carboxyl-PEGs, p-NP-PEGs, Tresyl-PEGs,aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs, maleimido-PEGs,hydroxyl-PEG-amine, amino-PEG-COOK hydroxyl-PEG-aldehyde, carboxylicanhydride type-PEG, functionalized PEG-phospholipid, and other similarand/or suitable reactive PEGs.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, at least 12 subunits, at least 13subunits, at least 14 subunits, at least 15 subunits, at least 16subunits, at least 17 subunits, at least 18 subunits, at least 19subunits, at least 20 subunits, at least 21 subunits, at least 22subunits, at least 23 subunits, or at least 24 subunits. In some suchembodiments, the PEG unit comprises no more than about 72 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, at least 12 subunits, at least 13subunits, at least 14 subunits, at least 15 subunits, at least 16subunits, at least 17 subunits, at least 18 subunits, at least 19subunits, at least 20 subunits, at least 21 subunits, at least 22subunits, at least 23 subunits, or at least 24 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, at least 12 subunits, at least 13subunits, at least 14 subunits, at least 15 subunits, at least 16subunits, at least 17 subunits, or at least 18 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, or at least 12 subunits.

In some embodiments, the PEG unit comprises at least 8 subunits, atleast 9 subunits, at least 10 subunits, at least 11 subunits, or atleast 12 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, or at least 8 subunits.

In some embodiments, the PEG unit comprises one or more linear PEGchains each having at least 2 subunits, at least 3 subunits, at least 4subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits,at least 8 subunits, at least 9 subunits, at least 10 subunits, at least11 subunits, at least 12 subunits, at least 13 subunits, at least 14subunits, at least 15 subunits, at least 16 subunits, at least 17subunits, at least 18 subunits, at least 19 subunits, at least 20subunits, at least 21 subunits, at least 22 subunits, at least 23subunits, or at least 24 subunits. In another embodiment, the PEG unitcomprises a combined total of at least 6 subunits, at least 8, at least10 subunits, or at least 12 subunits. In some such embodiments, the PEGunit comprises no more than a combined total of about 72 subunits,preferably no more than a combined total of about 36 subunits.

In some embodiments, the PEG unit comprises a combined total of from 4to 72, 4 to 60, 4 to 48, 4 to 36 or 4 to 24 subunits, from 5 to 72, 5 to60, 5 to 48, 5 to 36 or 5 to 24 subunits, from 6 to 72, 6 to 60, 6 to48, 6 to 36 or from 6 to 24 subunits, from 7 to 72, 7 to 60, 7 to 48, 7to 36 or 7 to 24 subunits, from 8 to 72, 8 to 60, 8 to 48, 8 to 36 or 8to 24 subunits, from 9 to 72, 9 to 60, 9 to 48, 9 to 36 or 9 to 24subunits, from 10 to 72, 10 to 60, 10 to 48, 10 to 36 or 10 to 24subunits, from 11 to 72, 11 to 60, 11 to 48, 11 to 36 or 11 to 24subunits, from 12 to 72, 12 to 60, 12 to 48, 12 to 36 or 12 to 24subunits, from 13 to 72, 13 to 60, 13 to 48, 13 to 36 or 13 to 24subunits, from 14 to 72, 14 to 60, 14 to 48, 14 to 36 or 14 to 24subunits, from 15 to 72, 15 to 60, 15 to 48, 15 to 36 or 15 to 24subunits, from 16 to 72, 16 to 60, 16 to 48, 16 to 36 or 16 to 24subunits, from 17 to 72, 17 to 60, 17 to 48, 17 to 36 or 17 to 24subunits, from 18 to 72, 18 to 60, 18 to 48, 18 to 36 or 18 to 24subunits, from 19 to 72, 19 to 60, 19 to 48, 19 to 36 or 19 to 24subunits, from 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24subunits, from 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24subunits, from 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24subunits, from 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24subunits, or from 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

In some embodiments, the PEG unit comprises one or more linear PEGchains having a combined total of from 4 to 72, 4 to 60, 4 to 48, 4 to36 or 4 to 24 subunits, from 5 to 72, 5 to 60, 5 to 48, 5 to 36 or 5 to24 subunits, from 6 to 72, 6 to 60, 6 to 48, 6 to 36 or 6 to 24subunits, from 7 to 72, 7 to 60, 7 to 48, 7 to 36 or 7 to 24 subunits,from 8 to 72, 8 to 60, 8 to 48, 8 to 36 or 8 to 24 subunits, from 9 to72, 9 to 60, 9 to 48, 9 to 36 or 9 to 24 subunits, from 10 to 72, 10 to60, 10 to 48, 10 to 36 or 10 to 24 subunits, from 11 to 72, 11 to 60, 11to 48, 11 to 36 or 11 to 24 subunits, from 12 to 72, 12 to 60, 12 to 48,12 to 36 or 12 to 24 subunits, from 13 to 72, 13 to 60, 13 to 48, 13 to36 or 13 to 24 subunits, from 14 to 72, 14 to 60, 14 to 48, 14 to 36 or14 to 24 subunits, from 15 to 72, 15 to 60, 15 to 48, 15 to 36 or 15 to24 subunits, from 16 to 72, 16 to 60, 16 to 48, 16 to 36 or 16 to 24subunits, from 17 to 72, 17 to 60, 17 to 48, 17 to 36 or 17 to 24subunits, from 18 to 72, 18 to 60, 18 to 48, 18 to 36 or 18 to 24subunits, from 19 to 72, 19 to 60, 19 to 48, 19 to 36 or 19 to 24subunits, from 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24subunits, from 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24subunits, from 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24subunits, from 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24subunits, or from 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

In some embodiments, the PEG unit is a derivatized linear single PEGchain having at least 2 subunits, at least 3 subunits, at least 4subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits,at least 8 subunits, at least 9 subunits, at least 10 subunits, at least11 subunits, at least 12 subunits, at least 13 subunits, at least 14subunits, at least 15 subunits, at least 16 subunits, at least 17subunits, at least 18 subunits, at least 19 subunits, at least 20subunits, at least 21 subunits, at least 22 subunits, at least 23subunits, or at least 24 subunits.

In some embodiments, the PEG unit is a derivatized linear single PEGchain having from 6 to 72, 6 to 60, 6 to 48, 6 to 36 or 6 to 24subunits, from 7 to 72, 7 to 60, 7 to 48, 7 to 36 or 7 to 24 subunits,from 8 to 72, 8 to 60, 8 to 48, 8 to 36 or 8 to 24 subunits, from 9 to72, 9 to 60, 9 to 48, 9 to 36 or 9 to 24 subunits, from 10 to 72, 10 to60, 10 to 48, 10 to 36 or 10 to 24 subunits, from 11 to 72, 11 to 60, 11to 48, 11 to 36 or 11 to 24 subunits, from 12 to 72, 12 to 60, 12 to 48,12 to 36 or 12 to 24 subunits, from 13 to 72, 13 to 60, 13 to 48, 13 to36 or 13 to 24 subunits, from 14 to 72, 14 to 60, 14 to 48, 14 to 36 or14 to 24 subunits, from 15 to 72, 15 to 60, 15 to 48, 15 to 36 or 15 to24 subunits, from 16 to 72, 16 to 60, 16 to 48, 16 to 36 or 16 to 24subunits, from 17 to 72, 17 to 60, 17 to 48, 17 to 36 or 17 to 24subunits, from 18 to 72, 18 to 60, 18 to 48, 18 to 36 or 18 to 24subunits, from 19 to 72, 19 to 60, 19 to 48, 19 to 36 or 19 to 24subunits, from 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24subunits, from 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24subunits, from 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24subunits, from 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24subunits, or from 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

In some embodiments, the PEG unit is a derivatized linear single PEGchain having from 2 to 72, 2 to 60, 2 to 48, 2 to 36 or 2 to 24subunits, from 2 to 72, 2 to 60, 2 to 48, 2 to 36 or 2 to 24 subunits,from 3 to 72, 3 to 60, 3 to 48, 3 to 36 or 3 to 24 subunits, from 3 to72, 3 to 60, 3 to 48, 3 to 36 or 3 to 24 subunits, from 4 to 72, 4 to60, 4 to 48, 4 to 36 or 4 to 24 subunits, from 5 to 72, 5 to 60, 5 to48, 5 to 36 or 5 to 24 subunits.

In some embodiments, a linear PEG unit is:

wherein;

the wavy line indicates site of attachment to the Multifunctional Linkeror M^(A) linker (e.g., to an amino acid in the M^(A) linker);

Y₇₁ is a PEG attachment unit;

Y₇₂ is a PEG capping unit;

Y₇₃ is an PEG coupling unit (i.e., for coupling multiple PEG subunitchains together);

d₉ is an integer from 2 to 72, preferably from 4 to 72, more preferablyfrom 6 to 72, from 8 to 72, from 10 to 72, from 12 to 72 or from 6 to24;

each d₁₀ is independently an integer from 1 to 72.

d₁₁ is an integer from 2 to 5.

In some embodiments, there are at least 6, preferably at least 8, atleast 10, or at least 12 PEG subunits in the PEG unit. In someembodiments, there are no more than 72 or 36 PEG subunits in the PEGunit.

In some embodiments, d₉ is 8 or about 8, 12 or about 12, 24 or about 24.

In some embodiments, each Y₇₂ is independently —C₁₋₁₀ alkyl, —C₂₋₁₀alkyl-CO₂H, —C₂₋₁₀ alkyl-OH, —C₂₋₁₀ alkyl-NH₂, —C₂₋₁₀ alkyl-NH(C₁₋₃alkyl), or C₂₋₁₀ alkyl-N(C₁₋₃ alkyl)₂.

In some embodiments, Y₇₂ is —C₁₋₁₀ alkyl, —C₂₋₁₀ alkyl-CO₂H, —C₂₋₁₀alkyl-OH, or —C₂₋₁₀ alkyl-NH₂.

The PEG coupling unit is part of the PEG unit and is non-PEG materialthat acts to connect two or more chains of repeating CH₂CH₂O— subunits.In some embodiments, the PEG coupling unit Y₇₃ is —C₂₋₁₀ alkyl-C(O)—NH—,—C₂₋₁₀ alkyl-NH—C(O)—, —C₂₋₁₀ alkyl-NH—, —C₂₋₁₀ alkyl-C(O)—, —C₂₋₁₀alkyl-O— or —C₂₋₄ alkyl-S—.

In some embodiments, each Y₇₃ is independently —C₁₋₁₀ alkyl-C(O)—NH—,—C₁₋₁₀ alkyl-NH—C(O)—, —C₂₋₁₀ alkyl-NH—, —C₂₋₁₀ alkyl-O—, —C₁₋₁₀alkyl-S—, or —C₁₋₁₀ alkyl-NH—.

The PEG attachment unit is part of the PEG unit and acts to link the PEGunit to the Multifunctional Linker or M^(A) linker (e.g., to an aminoacid in the M^(A) linker). In some embodiments, the amino acid has afunctional group that forms a bond with the PEG Unit. Functional groupsfor attachment of the PEG unit to the amino acid include sulfhydrylgroups to form disulfide bonds or thioether bonds, aldehyde, ketone, orhydrazine groups to form hydrazone bonds, hydroxylamine to form oximebonds, carboxylic or amino groups to form peptide bonds, carboxylic orhydroxy groups to form ester bonds, sulfonic acids to form sulfonamidebonds, alcohols to form carbamate bonds, and amines to form sulfonamidebonds or carbamate bonds or amide bonds. Accordingly, the PEG unit canbe attached to the amino acid, for example, via a disulfide, thioether,hydrazone, oxime, peptide, ester, sulfonamide, carbamate, or amide bond.Typically, the reaction for attaching the PEG unit can be acycloaddition, addition, addition/elimination or substitution reaction,or a combination thereof when applicable.

In some embodiments, the PEG attachment unit Y₇₁ is a bond, —C(O)—, —O—,—S—, —S(O)—, —S(O)₂—, —NR₅—, —C(O)O—, —C(O)—C₁₋₁₀ alkyl, —C(O)—C₁₋₁₀alkyl-O—, —C(O)—C₁₋₁₀ alkyl-CO₂—, —C(O)—C₁₋₁₀ alkyl-NR₅—, —C(O)—C₁₋₁₀alkyl-S—, —C(O)—C₁₋₁₀ alkyl-C(O)—NR₅—, —C(O)—C₁₋₁₀ alkyl-NR₅—C(O)—,—C₁₋₁₀ alkyl, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-CO₂—, —C₁₋₁₀ alkyl-NR₅—,—C₁₋₁₀ alkyl-S—, —C₁₋₁₀ alkyl-C(O)—NR₅—, —C₁₋₁₀ alkyl-NR₅—C(O)—,—CH₂CH₂SO₂—C₁₋₁₀ alkyl-, —CH₂C(O)—C₁₋₁₀ alkyl-, ═N—(O or N)—C₁₋₁₀alkyl-O—, ═N—(O or N)—C₁₋₁₀ alkyl-NR₅—, ═N—(O or N)—C₁₋₁₀ alkyl-CO₂—,═N—(O or N—C₁₋₁₀-alkyl-S—,

In some embodiments, Y₇₁ is —NH—, —C(O)—, a triazole group, —S—, or amaleimido-group such as

wherein the wavy line indicates attachment to the Multifunctional Linkeror M^(A) linker (e.g., to an amino acid in the M^(A) linker) and theasterisk indicates the site of attachment within the PEG Unit.

Examples of linear PEG units include, but are not limited to:

wherein the wavy line indicates site of attachment to the M^(A) linker(e.g., to an amino acid in the M^(A) linker), and each d₉ isindependently an integer from 4 to 24, 6 to 24, 8 to 24, 10 to 24, 12 to24, 14 to 24, or 16 to 24.

In some embodiments, d₉ is about 8, about 12, or about 24.

In some embodiments, the PEG unit is from about 300 daltons to about 5kilodaltons; from about 300 daltons, to about 4 kilodaltons; from about300 daltons, to about 3 kilodaltons; from about 300 daltons, to about 2kilodaltons; or from about 300 daltons, to about 1 kilodalton. In somesuch aspects, the PEG unit has at least 6 subunits or at least 8, 10 or12 subunits. In some embodiments, the PEG unit has at least 6 subunitsor at least 8, 10 or 12 subunits but no more than 72 subunits,preferably no more than 36 subunits.

Suitable polyethylene glycols may have a free hydroxy group at each endof the polymer molecule, or may have one hydroxy group etherified with alower alkyl, e.g., a methyl group. Also suitable for the practice of thedisclosure are derivatives of polyethylene glycols having esterifiablecarboxy groups. Polyethylene glycols are commercially available underthe trade name PEG, usually as mixtures of polymers characterized by anaverage molecular weight. Polyethylene glycols having an averagemolecular weight from about 300 to about 5000 are preferred, thosehaving an average molecular weight from about 600 to about 1000 beingparticularly preferred.

Other examples of hydrophilic groups that are suitable for theconjugates, scaffolds, and methods disclosed herein can be found ine.g., U.S. Pat. No. 8,367,065 column 13: U.S. Pat. No. 8,524,696 column6; WO2015/057699 and WO 2014/062697, the contents of each of which arehereby incorporated by reference in their entireties.

Antibody-Drug Conjugate (ADC) Type II

In some embodiments, the conjugate (e.g., the antibody-drug conjugate(ADC)) of the present disclosure is of Formula (III):PBRM-(A¹ _(a6)-L¹ _(s2)-L² _(y1)-D)_(d13)  (III)or pharmaceutically acceptable salt or solvate thereof, wherein:

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

A¹ is a stretcher unit;

a₆ is an integer 1 or 2;

L¹ is a specificity unit;

s₂ is an integer from about 0 to about 12;

L² is a spacer unit;

y₁ is an integer from 0 to 2; and

d₁₃ is an integer from about 1 to about 20.

In some embodiments, the conjugates of Formula (III) include those whereeach of the moieties defined for one of PBRM, D, A¹, a₆, L¹, s₂, L², y₁,and d₁₃ can be combined with any of the moieties defined for the othersof PBRM, D, A¹, a₆, L¹, s₂, L², y₁, and d₁₃.

In some embodiments, the conjugate (e.g., the antibody-drug conjugate(ADC)) of the present disclosure is of Formula (IIIa) or (IIIb):

or a pharmaceutically acceptable salt or solvate thereof,wherein:

PBRM denotes a protein based recognition-molecule;

each occurrence of D is independently a PBD drug moiety;

A¹ is a stretcher unit linked to the spacer unit L²;

a₆ is an integer 1 or 2;

L¹ is a specificity unit linked to the spacer unit L²;

s₆ is an integer from about 0 to about 12.

L² is a spacer unit;

y₁ is an integer 0, 1 or 2; and

d₁₃ is an integer from about 1 to about 20.

In some embodiments, the conjugates of any one of Formulae (IIIa)-(IIIb)include those where each of the moieties defined for one of PBRM, D, A¹,a₆, L¹, s₆, L², y₁, and d₁₃ can be combined with any of the moietiesdefined for the others of PBRM, D, A¹, a₆, L¹, s₆, L², y₁, and d₁₃.

In some embodiments, the conjugate (e.g., the antibody-drug conjugate(ADC)) of the present disclosure is of any one of Formulae (IIIc) to(IIIf):PBRM-(A¹ _(a6)-L¹ _(s2)-L² _(y1)-D)_(d13),  (IIIc)PBRM-(A¹ _(a6)-L¹ _(s2)-D)_(d13),  (IIId)PBRM-(A¹-L¹-D)_(d13),  (IIIe)PBRM-(A¹-D)_(d13), or  (IIIf)or a pharmaceutically acceptable salt or solvate thereof, wherein PBRM,A¹, a₆, L¹ s₂, L², y₁ D and d₁₃ are as defined herein.

In some embodiments, the conjugates of any one of Formulae (IIIc)-(IIIf)include those where each of the moieties defined for one of PBRM, A¹,a₆, L¹ s₂, L², y₁, D, and d₁₃ can be combined with any of the moietiesdefined for the others of PBRM, A¹, a₆, L¹ _(s2), L², y₁, D, and d₁₃.

In some embodiments, the PBRM specifically binds to a target molecule onthe surface of a target cell. An exemplary formula is:

wherein the asterisk indicates the point of attachment to the Drugmoiety (D), PRBM is targeting moiety, L¹ is a Specificity unit, A¹ is aStretcher unit connecting L to the PBRM, L² is a Spacer unit, which is acovalent bond, a self-immolative group or together with —OC(═O)— forms aself-immolative group, and L² is optional. —OC(═O)— may be considered asbeing part of L¹ or L², as appropriate.

In some embodiments, the PBRM specifically binds to a target molecule onthe surface of a target cell. An exemplary formula is:PBRM-A¹ _(a6)-L¹ _(s6)-L² _(y1)-*

wherein the asterisk indicates the point of attachment to the Drugmoiety (D), PBRM is the targeting moiety, L¹ is a Specificity unit, A¹is a Stretcher unit connecting L to the PBRM, L² is a Spacer unit whichis a covalent bond or a self-immolative group, and a₆ is an integer 1 or2, s₆ is an integer 0, 1 or 2, and y₁ is an integer 0, 1 or 2.

In the embodiments above, L can be a cleavable Specificity unit, and maybe referred to as a “trigger” that when cleaved activates aself-immolative group (or self-immolative groups) L², when aself-immolative group(s) is present. When the Specificity unit L¹ iscleaved, or the linkage (i.e., the covalent bond) between L¹ and L² iscleaved, the self-immolative group releases the PBD Drug moiety (D).

In some embodiments, the PBRM specifically binds to a target molecule onthe surface of a target cell. An exemplary formula is:

wherein the asterisk indicates the point of attachment to the PBD Drugmoiety (D), PBRM is the targeting moiety, L¹ is a Specificity unitconnected to L², A¹ is a Stretcher unit connecting L² to the PBRM, L² isa self-immolative group, and a₆ is an integer 1 or 2, s₆ is an integer0, 1 or 2, and y₁ is an integer 0, 1 or 2.

In the various embodiments discussed herein, the nature of L and L² canvary widely. These groups are chosen on the basis of theircharacteristics, which may be dictated in part, by the conditions at thesite to which the conjugate is delivered. Where the Specificity unit Ltis cleavable, the structure and/or sequence of L is selected such thatit is cleaved by the action of enzymes present at the target site (e.g.,the target cell). L¹ units that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. L¹ units that are cleavable under reducing oroxidizing conditions may also find use in the conjugates of the presentdisclosure.

In some embodiments, L¹ may comprise one amino acid or a contiguoussequence of amino acids. The amino acid sequence may be the targetsubstrate for an enzyme.

In some embodiments, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase. In someembodiments, L¹ may be cleaved by a lysosomal protease, such as, forexample, a cathepsin.

In some embodiments, L² is present and together with —C(═O)O— forms aself-immolative group or self-immolative groups. In some embodiments,—C(═O)O— also is a self-immolative group.

In some embodiments, where L¹ is cleavable by the action of an enzymeand L² is present, the enzyme cleaves the bond between L¹ and L²,whereby the self-immolative group(s) release the Drug moiety.

In some embodiments, L¹ and L², where present, may be connected by abond selected from: (i) —C(═O)NH: (ii) —C(═O)O—; (iii) —NHC(═O)—; (iv)—OC(═O)—; (v) —OC(═O)O—; (vi) —NHC(═O)O—; (vii) —OC(═O)NH—; (viii)—NHC(═O)NH—; and (ix) —O— (a glycosidic bond).

In some embodiments, an amino group of L¹ that connects to L² may be theN-terminus of an amino acid or may be derived from an amino group of anamino acid side chain, for example a lysine amino acid side chain.

In some embodiments, a carboxyl group of L¹ that connects to L² may bethe C-terminus of an amino acid or may be derived from a carboxyl groupof an amino acid side chain, for example a glutamic acid amino acid sidechain.

In some embodiments, a hydroxy group of L¹ that connects to L² may bederived from a hydroxy group of an amino acid side chain, such as, forexample, a serine amino acid side chain.

In some embodiments, —C(═O)O— and L² together form the group:

wherein the asterisk indicates the point of attachment to the Drugmoiety, the wavy line indicates the point of attachment to the L¹, Y₂ is—N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n₅ is an integer from 0 to 3.The phenylene ring is optionally substituted with one, two or threesubstituents as described herein.

In some embodiments, Y₂ is NH.

In some embodiments, n₅ is 0 or 1. Preferably, n₅ is 0.

In some embodiments, when Y₂ is NH and n₅ is 0, the self-immolativegroup may be referred to as a p-aminobenzylcarbonyl linker (PABC). Theself-immolative group will allow for release of the Drug moiety (i.e.,the PBD) when a remote site in the linker is activated, proceeding alongthe lines as shown below (for n₅=0):

wherein the asterisk indicates the attachment to the Drug, L³ is theactivated form of the remaining portion of the linker and the releasedDrug moiety is not shown. These groups have the advantage of separatingthe site of activation from the Drug.

In some embodiments, —C(═O)O— and L² together form a group selectedfrom:

wherein the asterisk, the wavy line, Y₂, and n₅ are as defined above.Each phenylene ring is optionally substituted with one, two or threesubstituents as described herein. In one embodiment, the phenylene ringhaving the Y₁ substituent is optionally substituted and the phenylenering not having the Y₁ substituent is unsubstituted.

In some embodiments, —C(═O)O— and L² together form a group selectedfrom:

wherein the asterisk, the wavy line, Y₂, and n₅ are as defined herein,Y₄ is O, S or NR, Y₃ is N, CH, or CR, and Y₅ is N, CH, or CR.

In some embodiments, Y₃ is N.

In some embodiments, Y₃ is CH.

In some embodiments, Y₄ is O or S.

In some embodiments, Y₅ is CH.

In some embodiments, the covalent bond between L¹ and L² is a cathepsinlabile (e.g., cleavable) bond.

In some embodiments, L¹ comprises a dipeptide. The amino acids in thedipeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the dipeptide comprises natural aminoacids. When the linker is a cathepsin labile linker, the dipeptide isthe site of action for cathepsin-mediated cleavage. The dipeptide thenis a recognition site for cathepsin.

In some embodiments, the group —X₅—X₆— in dipeptide, —NH—X₅—X₆—CO—, isselected from: (i) -Phe-Lys-; (ii) -Val-Ala; (iii) -Val-Lys-; (iv)-Ala-Lys; (v) -Ala-Ala; (vi) -Val-Cit; (vii) -Phe-Cit; (viii) -Leu-Cit;(ix) -Ile-Cit-Phe-Arg-, and (x) -Trp-Cit-; wherein Cit is citrulline. Insuch a dipeptide, —NH— is the amino group of X₅, and CO is the carbonylgroup of X₆.

In some embodiments, the group —X₅—X₆— in dipeptide, is selected from:(i) -Phe-Lys-, (ii) -Val-Ala-, (iii) -Ala-Ala-, (iv) -Val-Lys-, (v)-Ala-Lys-, and (vi) -Val-Cit-.

In some embodiments, the group —X₅—X₆— in dipeptide, is -Phe-Lys-,Val-Cit, -Ala-Ala- or -Val-Ala-.

Other dipeptide combinations of interest include: (i) -Gly-Gly-, (ii)-Pro-Pro-, and (iii) -Val-Glu-.

Other dipeptide combinations may be used, including those described byDubowchik et al., which is incorporated herein by reference.

In some embodiments, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below. Protected amino acid sequences are cleavable byenzymes. In some embodiments, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are amino acids having reactiveside chain functionality, such as, for example:

(i) Arg: Z, Mtr, Tos;

(ii) Asn: Trt, Xan;

(iii) Asp: Bzl, t-Bu;

(iv) Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;

(v) Glu: Bzl, t-Bu; Gin: Trt, Xan;

(vi) His: Boc, Dnp, Tos, Trt;

(vii) Lys: Boc, Z—CI, Fmoc, Z;

(viii) Ser: Bzl, TBDMS, TBDPS;

(ix) Thr: Bz;

(x) Trp: Boc; or

(xi) Tyr: Bzl, Z, Z—Br.

In some embodiments, —X₆— is connected indirectly to the Drug moiety. Insuch an embodiment, the Spacer unit L₂ is present.

In some embodiments, the dipeptide is used in combination with aself-immolative group(s) (the Spacer unit). The self-immolative group(s)may be connected to —X₆—.

When a self-immolative group is present, —X₆— is connected directly tothe self-immolative group. In one embodiment, —X₆— is connected to thegroup Y₂ of the self-immolative group. Preferably the group —X₆—CO— isconnected to Y₂, wherein Y₂ is NH.

In some embodiments, —X₅ is connected directly to A¹. Preferably thegroup NH—X₅— (the amino terminus of X₅) is connected to A¹. A¹ maycomprise the functionality —CO— thereby to form an amide link with —X₅.

In some embodiments, L¹ and L² together with —OC(═O)— comprise the group—X₅—X₆-PABC-. The PABC group is connected directly to the Drug moiety.In one example, the self-immolative group and the dipeptide togetherform the group -Phe-Lys-PABC-, is:

wherein the asterisk indicates the point of attachment to the Drugmoiety, and the wavy line indicates the point of attachment to theremaining portion of L¹ or the point of attachment to A. In someembodiments, the wavy line indicates the point of attachment to A¹.

In some embodiments, the self-immolative group and the dipeptidetogether form the group -Val-Ala-PABC- or -Ala-Ala-PABC are;

wherein the asterisk and the wavy line are as defined above.

In some embodiments, L¹ and L² together with —OC(═O)— are:

wherein the asterisk indicates the point of attachment to the Drugmoiety, the wavy line indicates the point of attachment to A¹, Y₂ is acovalent bond or a functional group, and Y₆ is a group that issusceptible to cleavage thereby to activate a self-immolative group.

In some embodiments, Y₆ is selected such that the group is susceptibleto cleavage, e.g., by light or by the action of an enzyme. In someembodiments, Y₆ may be —NO₂ or glucuronic acid (e.g., p-glucuronicacid). The former may be susceptible to the action of a nitroreductase,the latter to the action of a β-glucuronidase.

In some embodiments, the group Y₂ may be a covalent bond.

In some embodiments, the group Y₂ may be a functional group selectedfrom (i) —C(═O)—; (ii) —NH—; (iii) —O—; (iv) —C(═O)NH—; (v) —C(═O)O—;(vi) —NHC(═O)—; (vii) —OC(═O)—; (viii) —OC(═O)O—; (ix) —NHC(═O)O—; (x)—OC(═O)NH—; (xi) —NHC(═O)NH—; (xii) —NHC(═O)NH; (xiii) —C(═O)NHC(═O)—;(xiv) SO₂; and (v) —S—.

In some embodiments, the group Y₂ is preferably —NH—, —CH₂—, —O—, and—S—.

In some embodiments, L¹ and L² together with —OC(═O)— is:

wherein the asterisk indicates the point of attachment to the Drugmoiety, the wavy line indicates the point of attachment to A¹, Y₂ is acovalent bond or a functional group and Y₆ is glucuronic acid (e.g.,β-glucuronic acid). Y₂ is preferably a functional group selected from—NH—.

In some embodiments, L¹ and L² together are:

wherein the asterisk indicates the point of attachment to the remainderof L² or the Drug moiety, the wavy line indicates the point ofattachment to A¹, Y₂ is a covalent bond or a functional group and Y₆ isglucuronic acid (e.g., β-glucuronic acid). Y₂ is preferably a functionalgroup selected from —NH—, —CH₂—, —O—, and —S—.

In some embodiments, Y₂ is a functional group as set forth above, thefunctional group is linked to an amino acid, and the amino acid islinked to the Stretcher unit A. In some embodiments, amino acid isβ-alanine. In such an embodiment, the amino acid is equivalentlyconsidered part of the Stretcher unit.

In some embodiments, the Specificity unit L¹ and the PBRM are indirectlyconnected via the Stretcher unit.

In some embodiments, L¹ and A¹ may be connected by a bond selected from:(i) —C(═O)NH—; (ii) —C(═O)O—; (iii) —NHC(═O)—; (iv) —OC(═O)—; (v)—OC(═O)O—; (vi) —NHC(═O)O—; (vii) —OC(═O)NH—; and (viii) —NHC(═O)NH—.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PRBM moiety, and b₁ is aninteger from 0 to 6. In one embodiment, b₁ is 5.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PRBM moiety, and b₁ is aninteger from 0 to 6. In one embodiment, b₁ is 5.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PBRM moiety, and b₁ is aninteger from 0 to 6. In one embodiment, b₁ is 5.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PBRM moiety, and b₁ is aninteger from 0 to 6. In one embodiment, b₁ is 5.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the group A¹ is:

wherein the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the connection between the PBRM moiety and A¹ isthrough a thiol residue of the PBRM moiety and a maleimide group of A.

In some embodiments, the connection between the PBRM moiety and A¹ is:

wherein the asterisk indicates the point of attachment to the remainingportion of A¹, L¹, L² or D, and the wavy line indicates the point ofattachment to the remaining portion of the PBRM moiety. In thisembodiment, the S atom is typically derived from the PBRM moiety.

In each of the embodiments above, an alternative functionality may beused in place of the malemide-derived group is:

wherein the wavy line indicates the point of attachment to the PBRMmoiety as before, and the asterisk indicates the bond to the remainingportion of the A¹ group, or to L¹, L² or D.

In some embodiments, the maleimide-derived group is replaced with thegroup:

wherein the wavy line indicates point of attachment to the PBRM moiety,and the asterisk indicates the bond to the remaining portion of the Agroup, or to L¹, L² or D.

In some embodiments, the maleimide-derived group is replaced with agroup, which optionally together with a PBRM moiety (e.g., a PBRM), isselected from: (i) —C(═O)NH—; (ii) —C(═O)O—; (iii) —NHC(═O)—; (iv)—OC(═O)—; (v) —OC(═O)O—; (vi) —NHC(═O)O—; (vii) —OC(═O)NH—; (viii)—NHC(═O)NH—; (ix) —NHC(═O)NH; (x) —C(═O)NHC(═O)—; (xi) —S—; (xii) —S—S—;(xiii) —CH₂C(═O)—; (xiv) —C(═O)CH₂—; (xv)=N—NH—; and (xvi) —NH—N═. Ofthese —C(═O)CH₂— may be preferred especially when the carbonyl group isbound to —NH—.

In some embodiments, the maleimide-derived group is replaced with agroup, which optionally together with the PBRM moiety, is selected from:

wherein the wavy line indicates either the point of attachment to themoiety or the bond to the remaining portion of the A¹ group, and theasterisk indicates the other of the point of attachment to the PBRMmoiety or the bond to the remaining portion of the A¹ group.

Other groups suitable for connecting L¹ to the PBRM are described in WO2005/082023.

In some embodiments, the Stretcher unit A¹ is present, the Specificityunit L¹ is present and Spacer unit L² is absent. Thus, L¹ and the Drugmoiety are directly connected via a bond.

Equivalently in this embodiment, L² is a bond.

In some embodiments, L¹ and D may be connected by a bond selected from:(i) —C(═O)N<; (ii) —C(═O)O—; (iii) —NHC(═O)—; (iv) —OC(═O)—; (v)—OC(═O)O—; (vi) —NHC(═O)O; (vii) —OC(═O)N<; and (viii) —NHC(═O)N<;wherein N< or O— are part of D.

In some embodiments, L¹ and D are preferably connected by a bondselected from: —C(═O)N<, and —NHC(═O)—.

In some embodiments, L¹ comprises a dipeptide and one end of thedipeptide is linked to D. As described above, the amino acids in thedipeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the dipeptide comprises natural aminoacids. Where the linker is a cathepsin labile linker, the dipeptide isthe site of action for cathepsin-mediated cleavage. The dipeptide thenis a recognition site for cathepsin.

In some embodiments, the group —X₅—X₆— in dipeptide, —NH—X₅—X₆—CO—, isselected from: (i) -Phe-Lys-; (ii) -Val-Ala-; (iii) -Ala-Ala-; (iv)-Val-Lys-; (v) -Ala-Lys-; (vi) -Val-Cit-; (vii) -Phe-Cit-; (viii)-Leu-Cit-; (ix) -lie-Cit-; (x) -Phe-Arg-; and (xi) -Trp-Cit-; whereinCit is citrulline. In such a dipeptide, —NH— is the amino group of X₅,and CO is the carbonyl group of X₆.

In some embodiments, the group —X₅—X₆— in dipeptide, —NH—X₅—X₆—CO—, isselected from: (i) -Phe-Lys-; (ii) -Val-Ala-; (iii) -Ala-Ala-; (iv)-Val-Lys-; (v) -Ala-Lys-; and (vi) -Val-Cit-.

In some embodiments, the group —X X2- in dipeptide, is -Phe-Lys-,-Ala-Ala- or -Val-Ala-.

Other dipeptide combinations of interest include: (i) -Gly-Gly-; (ii)-Pro-Pro-; and (iii) -Val-Glu-.

Other dipeptide combinations may be used, including those describedabove.

In some embodiments, L-D is:—NH—X₅—X₆—CO—N<*wherein —NH—X₅—X₆—CO— is the dipeptide, —N< is part of the Drug moiety,the asterisk indicates the points of attachment to the remainder of theDrug moiety, and the wavy line indicates the point of attachment to theremaining portion of L¹ or the point of attachment to A. Preferably, thewavy line indicates the point of attachment to A¹.

In some embodiments, the dipeptide is valine-alanine and L¹-D is:

wherein the asterisks, —N< and the wavy line are as defined above.

In some embodiments, the dipeptide is alanine-alanine and L-D is:

wherein the asterisks, —N< and the wavy line are as defined above.

In some embodiments, the dipeptide is phenylalanine-lysine and L¹-D is:

wherein the asterisks, —N< and the wavy line are as defined above.

In some embodiments, the dipeptide is valine-citrulline.

In some embodiments, the groups A -L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, and b₁is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, and b₁is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 7, preferably 3 to 7, mostpreferably 3 or 7. one embodiment, the groups A1

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, and b₁is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L or D, thewavy line indicates the point of attachment to the PBRM moiety, and b₁is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the groups A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, thewavy line indicates the point of attachment to the PBRM moiety, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, mostpreferably 4 or 8.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the rest of the PBRM moiety, and b₁ is an integer from 0to 6. In some embodiments, b₁ is 5.

In some embodiments, the group PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, and b₁ is an integerfrom 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, n₆ is an integer 0 or 1,and n₇ is an integer from 0 to 30. In a preferred embodiment, n₆ is 1and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, n₆ is an integer 0 or 1,and n₇ is an integer from 0 to 30. In a preferred embodiment, n₆ is 1and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, b₁ is an integer from 0to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, b₁ is an integer from 0to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, b₁ is an integer from 0to 6. In some embodiments, b₁ is 5.

In some embodiments, the groups PBRM-A¹-L¹ are:

wherein the asterisk indicates the point of attachment to L² or D, S isa sulfur group of the PBRM moiety, the wavy line indicates the point ofattachment to the remainder of the PBRM moiety, b₁ is an integer from 0to 6. In some embodiments, b₁ is 5.

In some embodiments, the Stretcher unit is an acetamide unit, having theformula:

wherein the asterisk indicates the point of attachment to the remainderof the Stretcher unit, L¹ or D, and the wavy line indicates the point ofattachment to the PBRM moiety.Linker-Drugs

In other embodiments, Linker-Drug compounds are provided for conjugationto a PBRM moiety. In some embodiments, the Linker-Drug compounds aredesigned for connection to a PBRM.

In some embodiments, the Drug Linker is

wherein the asterisk indicates the point of attachment to the Drugmoiety (D, as defined above), A² is a Stretcher group (A¹) to form aconnection to a PBRM moiety, L¹ is a Specificity unit, L² (a Spacerunit) is a covalent bond or together with —OC(═O)— forms aself-immolative group(s).

In another embodiment, the Drug Linker compound isA²-L¹-L²-wherein the asterisk indicates the point of attachment to the Drugmoiety (D), A² is a Stretcher unit (A1) to form a connection to a PBRMmoiety, L¹ is a Specificity unit, L² (a Spacer unit) is a covalent bondor a self-immolative group(s).

L¹ and L² are as defined above. References to connection to A¹ can beconstrued here as referring to a connection to A².

In some embodiments, where L¹ comprises an amino acid, the side chain ofthat amino acid may be protected. Any suitable protecting group may beused. In some embodiments, the side chain protecting groups areremovable with other protecting groups in the compound, where present.In other embodiments, the protecting groups may be orthogonal to otherprotecting groups in the molecule, where present.

Suitable protecting groups for amino acid side chains include thosegroups described in the Novabiochem Catalog 2006/2007. Protecting groupsfor use in a cathepsin labile linker are also discussed in Dubowchik etal.

In certain embodiments, the group L¹ includes a Lys amino acid residue.The side chain of this amino acid may be protected with a Boc or Allocprotected group. A Boc protecting group is most preferred.

The functional group A² forms a connecting group upon reaction with aPBRM moiety.

In some embodiments, the functional group A² is or comprises an amino,carboxylic acid, hydroxy, thiol, or maleimide group for reaction with anappropriate group on the PBRM moiety.

In a preferred embodiment, A² comprises a maleimide group.

In some embodiments, the group A² is an alkyl maleimide group. Thisgroup is suitable for reaction with thiol groups, particularly cysteinethiol groups, present in the PBRM, for example present in an antibody.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, L² or D,and b₁ is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, L² or D,and b₁ is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n, is 0 to 10, 1 to 2, preferably 4 to 8, andmost preferably 4 or 8.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, n₆ is aninteger 0 or 1, and 7 is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, andmost preferably 4 or 8.

In some embodiments, the group A²:

wherein the asterisk indicates the point of attachment to L¹, L² or D,and b₁ is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, L² or D,and b₁ is an integer from 0 to 6. In some embodiments, b₁ is 5.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 2 preferably 4 to 8, andmost preferably 4 or 8.

In some embodiments, the group A² is:

wherein the asterisk indicates the point of attachment to L¹, n₆ is aninteger 0 or 1, and n₇ is an integer from 0 to 30. In a preferredembodiment, n₆ is 1 and n₇ is 0 to 10, 1 to 8, preferably 4 to 8, andmost preferably 4 or 8.

In each of the embodiments above, an alternative functionality may beused in place of the malemide group shown below:

wherein the asterisk indicates the bond to the remaining portion of theA² group.

In some embodiments, the maleimide-derived group is replaced with thegroup:

wherein the asterisk indicates the bond to the remaining portion of theA² group.

In some embodiments, the maleimide group is replaced with a groupselected from: (i) —C(═O)OH; (ii) —OH; (iii) —NH₂; (iv) —SH; (v)—C(═O)CH₂X; wherein X₇ is CI, Br or I; (vi) —CHO; (vii) —C═CH; and(viii) —N₃ (azide). Of these, —C(═O)CH₂X₇ may be preferred, especiallywhen the carbonyl group is bound to —NH—.

In some embodiments, L¹ is present, and A² is —NH₂, —NHMe, —COOH, —OH or—SH.

In some embodiments, where L¹ is present, A² is —NH₂ or —NHMe. Eithergroup may be the N-terminal of an L¹ amino acid sequence.

In some embodiments, L¹ is present and A² is —NH₂, and L¹ is an aminoacid sequence —X₅—X₆—, as defined above.

In some embodiments, L¹ is present and A² is COOH. This group may be theC-terminal of an L¹ amino acid sequence.

In some embodiments, L¹ is present and A² is OH.

In some embodiments, L¹ is present and A² is SH.

The group A² may be convertible from one functional group to another. Inone embodiment, L¹ is present and A² is —NH₂. This group is convertibleto another group A² comprising a maleimide group. In some embodiments,the group —NH₂ may be reacted with an acids or an activated acid (e.g.,N-succinimide forms) of those A² groups comprising maleimide shownabove.

The group A² may therefore be converted to a functional group that ismore appropriate for reaction with a PBRM moiety.

As noted above, In some embodiments, L¹ is present and A² is —NH₂,—NHMe, —COOH, —OH or —SH. In a further embodiment, these groups areprovided in a chemically protected form. The chemically protected formis therefore a precursor to the linker that is provided with afunctional group.

In some embodiments, A² is —NH₂ in a chemically protected form. Thegroup may be protected with a carbamate protecting group. The carbamateprotecting group may be selected from the group consisting of: Alloc,Fmoc, Boc, Troc, Teoc, Cbz and PNZ.

Preferably, where A² is —NH₂, it is protected with an Alloc or Fmocgroup.

In some embodiments, where A² is —NH₂, it is protected with an Fmocgroup.

In some embodiments, the protecting group is the same as the carbamateprotecting group of the capping group.

In some embodiments, the protecting group is not the same as thecarbamate protecting group of the capping group. In this embodiment, itis preferred that the protecting group is removable under conditionsthat do not remove the carbamate protecting group of the capping group.

The chemical protecting group may be removed to provide a functionalgroup to form a connection to a PBRM moiety. Optionally, this functionalgroup may then be converted to another functional group as describedabove.

In some embodiments, the active group is an amine. This amine ispreferably the N-terminal amine of a peptide, and may be the N-terminalamine of the preferred dipeptides of the present disclosure. The activegroup may be reacted to yield the functional group that is intended toform a connection to a PBRM moiety.

In other embodiments, the Linker unit is a precursor to the Linker unithaving an active group. In this embodiment, the Linker unit comprisesthe active group, which is protected by way of a protecting group. Theprotecting group may be removed to provide the Linker unit having anactive group.

Where the active group is an amine, the protecting group may be an amineprotecting group, such as those described in Green and Wuts. Theprotecting group is preferably orthogonal to other protecting groups,where present, the Linker unit.

In some embodiments, the protecting group is orthogonal to the cappinggroup. Thus, the active group protecting group is removable whilstretaining the capping group. In other embodiments, the protecting groupand the capping group is removable under the same conditions as thoseused to remove the capping group.

In some embodiments, the Linker unit is:

wherein the asterisk indicates the point of attachment to the Drugmoiety, and the wavy line indicates the point of attachment to theremaining portion of the Linker unit, as applicable or the point ofattachment to A². Preferably, the wavy line indicates the point ofattachment to A.

In some embodiments, the Linker unit is:

wherein the asterisk and the way line are as defined above.

Other functional groups suitable for use in forming a connection betweenL and the PBRM are described in WO 2005/082023.

Protein-Based Recognition Molecules (PBRMs)

The protein-based recognition molecule directs the conjugates comprisinga peptide linker to specific tissues, cells, or locations in a cell. Theprotein-based recognition molecule can direct the conjugate in cultureor in a whole organism, or both. In each case, the protein-basedrecognition molecule has a ligand that is present on the cell surface ofthe targeted cell(s) to which it binds with an effective specificity,affinity and avidity. In some embodiments, the protein-based recognitionmolecule targets the conjugate to tissues other than the liver. In otherembodiments the protein-based recognition molecule targets the conjugateto a specific tissue such as the liver, kidney, lung or pancreas. Theprotein-based recognition molecule can target the conjugate to a targetcell such as a cancer cell, such as a receptor expressed on a cell suchas a cancer cell, a matrix tissue, or a protein associated with cancersuch as tumor antigen. Alternatively, cells comprising the tumorvasculature may be targeted. Protein-based recognition molecules candirect the conjugate to specific types of cells such as specifictargeting to hepatocytes in the liver as opposed to Kupffer cells. Inother cases, protein-based recognition molecules can direct theconjugate to cells of the reticular endothelial or lymphatic system, orto professional phagocytic cells such as macrophages or eosinophils. (Insuch cases the conjugate itself might also be an effective deliverysystem, without the need for specific targeting).

In still other embodiments, the protein based recognition molecule cantarget the conjugate to a location within the cell, such as the nucleus,the cytoplasm, or the endosome, for example. In specific embodiments,the protein based recognition molecule can enhance cellular binding toreceptors, or cytoplasmic transport to the nucleus and nuclear entry orrelease from endosomes or other intracellular vesicles.

In specific embodiments the protein based recognition molecules includeantibodies, proteins and peptides or peptide mimics.

In a preferred embodiment, the protein based recognition moleculecomprises a sulfhydryl group and the protein based recognition moleculeis conjugated to the Linker-Drug moiety by forming a covalent bond viathe sulfhydryl group and a functional group of the Linker-Drug moiety.

Exemplary antibodies or antibodies derived from Fab, Fab2, scFv or camelantibody heavy-chain fragments specific to the cell surface markers,include, but are not limited to, 5T4, AOC3, ALK, AXL, C242, C4.4a,CA-125, CCL11, CCR 5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19,CA19-9, CDH6, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37,CD38, CD40, CD41, CD44, CD44 v6, CD51, CD52, CD54, CD56, CD62E, CD62P,CD62L, CD70, CD74, CD79-B, CD80, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM-5, clumping factor, CTLA-4, CXCR2, EGFR (HER1),ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FGFR (i.e. FGFR1, FGFR2,FGFR3, FGFR4), FLT3, folate receptor, FAP, GD2, GD3, GPNMB, GCC(GUCY2C), HGF, HER2, HER3, HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1,EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP,adrenergic receptor-beta2, Claudine 3, LIV1, LY6E, Mesothelin, MUC1,MUC13, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2,PTK7, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor,TROP-2, frizzled-7, integrins (including α₄, α_(v)β₃, α_(v)β₅, α_(v)β₆,α₁β₄, α₄β₁, α₄β₇, α₅β₁, α₆β₄, α_(IIb)β₃ integrins), IFN-α, IFN-γ, IgE,IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6,interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin (CD62L),mucin, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostaticcarcinoma cell, Pseudomonas aeruginosa, rabies, RANKL, respiratorysyncytial virus, Rhesus factor, SLAMF7, sphingosine-1-phosphate, TAG-72,T-cell receptor, tenascin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1,TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, and thelike.

In some embodiments, the antibodies or antibody derived from Fab, Fab2,scFv or camel antibody heavy-chain fragments specific to the cellsurface markers include CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31,CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70,CD138, CD141, CD326, CEA, CTLA-4, EGFR (HER1), ErbB2, ErbB3, FAP, folatereceptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1,EphA2, EpCAM, 5T4, TAG-72, tenascin C, TRPV1, CFTR, gpNMB, CA9, Cripto,ACE, APP, PDGFR α, phosphatidylserine, prostatic carcinoma cells,adrenergic receptor-beta2, Claudine 3, mucin, MUC1, NaPi2b, B7H3, B7H4,C4.4a, CEACAM-5, MUC13, TROP-2, frizzled-7, Mesothelin, IL-2 receptor,IL-4 receptor, IL-13 receptor and integrins (including α_(v)β₃, α_(v)β₅,α_(v)β₆, α₁β₄, α₄β₁, α₅β₁, α₆β₄ integrins), tenascin C, TRAIL-R2 andvimentin.

Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO),adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab,ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab,anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), aselizumab, atlizumab(tocilizumab, Actemra, RoActemra), atorolimumab, bapineuzumab,basiliximab (Simulect), bavituximab, bectumomab (LYMPHOSCAN), belimumab(BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN),bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab,blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS),cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab,certolizumab, cetuximab (ERBITUX), citatuzumab, cixutumumab,clenoliximab, clivatuzumab, conatumumab, CR6261, dacetuzumab, daclizumab(ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab,dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edobacomab, edrecolomab(PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab,elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab(REXOMUN), etaracizumab (ABEGRIN), exbivirumab, fanolesomab(NEUTROSPEC), faralimomab, farletuzumab, fevizumab, fezakinumab,figitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab,gantenerumab, gavilimomab, gemtuzumab, girentuximab, glembatumumab,golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab(INDIMACIS-125), imciromab (MYOSCINT), infliximab (REMICADE),intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, keliximab,labetuzumab (CEA-CIDE), lebrikizumab, lemalesomab, lerdelimumab,lexatumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab,mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab,milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAX),muromonab-CD3 (ORTHOCLONE OKT3), nacolomab, naptumomab, natalizumab(TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM),nofetumomab, ocrelizumab, odulimomab, ofatumumab (ARZERRA), olaratumab,omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX),otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab(VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab(OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO 140,rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab,regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), robatumumab,rontalizumab, rovelizumab (LEUKARREST), ruplizumab (ANTOVA), satumomabpendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab,solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab(LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab,tanezumab, taplitumomab paptox, tefibazumab (AUREXIS), telimomab,tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab(tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA),toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN),tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab(STELERA), vapaliximab, vedolizumab, veltuzumab, vepalimomab,visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab(HuMEX-EGFr), zanolimumab (HuMAX-CD4), ziralimumab and zolimomab.

In some embodiments, the antibodies are directed to cell surface markersfor 5T4, CA-125, CEA, CDH6, CD3, CD19, CD20, CD22, CD30, CD33, CD40,CD44, CD51, CTLA-4, CEACAM5, EpCAM, HER2, EGFR (HER1), FAP, folatereceptor, GCC (GUCY2C), HGF, integrin α_(v)β₃, integrin α₅β₁, IGF-1receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, PTK7,phosphatidylserine, prostatic carcinoma cells, PDGFR α, TAG-72, tenascinC, TRAIL-R2, VEGF-A and VEGFR2. In this embodiment the antibodies areabagovomab, adecatumumab, alacizumab, altumomab, anatumomab,arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab,blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab,citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab,epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab,gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab,inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab,matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab,oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab(RITUXAN), rilotumumab, robatumumab, satumomab, sibrotuzumab,taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab),tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab,tucotuzumab celmoleukin, volociximab and zalutumumab.

In specific embodiments the antibodies directed to cell surface markersfor HER2 are pertuzumab or trastuzumab and for EGFR (HER1) the antibodyis cetuximab or panitumumab; and for CD20 the antibody is rituximab andfor VEGF-A is bevacizumab and for CD-22 the antibody is epratuzumab orveltuzumab and for CEA the antibody is labetuzumab.

Exemplary peptides or peptide mimics include integrin targeting peptides(RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptortargeting peptides, prostate specific membrane bound antigen (PSMA)targeting peptides, lipoprotein receptor LRPI targeting, ApoE proteinderived peptides, ApoA protein peptides, somatostatin receptor targetingpeptides, chlorotoxin derived peptides, and bombesin.

In specific embodiments the peptides or peptide mimics are LHRH receptortargeting peptides and ErbB2 (HER2) receptor targeting peptides

Exemplary proteins comprise insulin, transferrin, fibrinogen-gammafragment, thrombospondin, claudin, apolipoprotein E, Affibody moleculessuch as, for example, ABY-025, Ankyrin repeat proteins, ankyrin-likerepeats proteins and synthetic peptides.

In some embodiments, the protein-drug conjugates comprise broad spectrumcytotoxins in combination with cell surface markers for HER2 such aspertuzumab or trastuzumab; for EGFR such as cetuximab and panitumumab;for CEA such as labetuzumab; for CD20 such as rituximab; for VEGF-A suchas bevacizumab; or for CD-22 such as epratuzumab or veltuzumab.

In other embodiments, the protein-drug conjugates or protein conjugatesused in the disclosure comprise combinations of two or more proteinbased recognition molecules, such as, for example, combination ofbispecific antibodies directed to the EGF receptor (EGFR) on tumor cellsand to CD3 and CD28 on T cells; combination of antibodies or antibodyderived from Fab, Fab2, scFv or camel antibody heavy-chain fragments andpeptides or peptide mimetics; combination of antibodies or antibodyderived from Fab, Fab2, scFv or camel antibody heavy-chain fragments andproteins; combination of two bispecific antibodies such as CD3×CD19 plusCD28×CD22 bispecific antibodies.

In other embodiments, the protein-drug conjugates or protein conjugatesused in the disclosure comprise protein based recognition molecules areantibodies against antigens, such as, for example, Trastuzumab,Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab, Labetuzumab,B7-H4, B7-H3, CA125, CDH6, CD33, CXCR2, CEACAM5, EGFR, FGFR1, FGFR2,FGFR3, FGFR4, GCC (GUCY2C), HER2, LIV1, LY6E, NaPi2b, c-Met, mesothelin,NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, PTK7, c-Kit, MUC1, MUC13. and5T4.

In a specific embodiment, the protein-drug conjugates or proteinconjugates of the disclosure comprise protein based recognitionmolecules which are antibodies against 5T4, such as, for example ahumanized anti-5T4 scFvFc antibody.

Examples of suitable 5T4 targeting ligands or immunoglobulins includethose which are commercially available, or have been described in thepatent or non-patent literature, e.g., U.S. Pat. Nos. 8,044,178,8,309,094, 7,514,546, EP1036091 (commercially available as TroVax™,Oxford Biomedica), EP2368914A1, WO 2013041687 A1 (Amgen), US2010/0173382, and P. Sapra, et al., Mol. Cancer Ther. 2013, 12:38-47. Ananti-5T4 antibody is disclosed in U.S. Provisional Application No.61/877,439, filed Sep. 13, 2013 and U.S. Provisional Application No.61/835,858, filed Jun. 17, 2013. The contents of each of the patentdocuments and scientific publications are herein incorporated byreference in their entireties.

As used herein, the term “5T4 antigen-binding portion” refers to apolypeptide sequence capable of selectively binding to a 5T4 antigen. Inexemplary conjugates, the 5T4 antigen-binding portion generallycomprises a single chain scFv-Fc form engineered from an anti-5T4antibody. A single-chain variable fragment (scFv-Fc) is a fusion proteinof the variable regions of the heavy (VH) and light chains (VL) of animmunoglobulin, connected with a linker peptide, and further connectedto an Fc region comprising a hinge region and CH₂ and CH₃ regions of anantibody (any such combinations of antibody portions with each other orwith other peptide sequences is sometimes referred to herein as an“immunofusion” molecule). Within such a scFvFc molecule, the scFvsection may be C-terminally linked to the N-terminus of the Fc sectionby a linker peptide.

In other specific embodiments, the protein-drug conjugates or proteinconjugates of the disclosure comprise protein based recognitionmolecules which are Her-2 or NaPi2b antibodies.

In some embodiments, the Her-2 antibody suitable for the conjugate orscaffold of the disclosure comprises a variable heavy chaincomplementarity determining region 1 (CDRH1) comprising the amino acidsequence FTFSSYSMN (SEQ ID NO: 25); a variable heavy chaincomplementarity determining region 2 (CDRH2) comprising the amino acidsequence YISSSSSTIYYADSVKG (SEQ ID NO: 26); a variable heavy chaincomplementarity determining region 3 (CDRH3) comprising the amino acidsequence GGHGYFDL (SEQ ID NO: 27); a variable light chaincomplementarity determining region 1 (CDRL1) comprising the amino acidsequence RASQSVSSSYLA (SEQ ID NO: 28); a variable light chaincomplementarity determining region 2 (CDRL2) comprising the amino acidsequence GASSRAT (SEQ ID NO: 21); and a variable light chaincomplementarity determining region 3 (CDRL3) comprising the amino acidsequence QQYHHSPLT (SEQ ID NO: 29) (see, e.g., US20150366987(A1)published Dec. 24, 2015). In some embodiments, the NaPi2b antibodysuitable for the conjugate or scaffold of the disclosure comprises avariable light chain complementarity determining region 1 (CDRL1)comprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8); avariable light chain complementarity determining region 2 (CDRL2)comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9); a variablelight chain complementarity determining region 3 (CDRL3) comprising theamino acid sequence QQYSKLPLT (SEQ ID NO: 10); a variable heavy chaincomplementarity determining region 1 (CDRH1) comprising the amino acidsequence GYTFTGYNIH (SEQ ID NO: 5); a variable heavy chaincomplementarity determining region 2 (CDRH2) comprising the amino acidsequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); and a variable heavy chaincomplementarity determining region 3 (CDRH3) comprising the amino acidsequence GETARATFAY (SEQ ID NO: 7)(see, e.g., co-pending applicationU.S. Ser. No. 15/457,574 filed Mar. 13, 2017).

PBD Drug Moiety (D)

In some embodiments, the PBD drug moiety (D) is of Formula (IV),

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer, wherein:

E″ is a direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment), E or

in which

denotes direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment) via a functional group of E;

D″ is D′ or

in which

denotes direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment) via a functional group of D′;

R″₇ is a direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment), R or

in which

denotes direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment) via a functional group of R₇;

R″₁₀ is a direct or indirect linkage to the PBRM (e.g., antibody orantibody fragment), R₁₀

or

in which

denotes direct or indirect linkage the PBRM (e.g., antibody or antibodyfragment) via a functional group of R₁₀; and wherein the PBD drug moiety(D) is directly or indirectly linked to the PBRM (e.g., antibody orantibody fragment) via a functional group of one of E″, D″, R″₇, andR″₁₀.

In some embodiments, E″ is direct or indirect linkage to L^(C), E, or

in which

denotes direct or indirect linkage to L^(C) via a functional group of E.

In some embodiments, E″ is a direct or indirect linkage to L^(D), E, or

in which

denotes direct or indirect linkage to L^(D) via a functional group of E.

In some embodiments, D″ is D′ or

in which

denotes direct or indirect linkage to L^(C) via a functional group ofD′.

In some embodiments, D″ is D′ or

in which

denotes direct or indirect linkage to L^(D) via a functional group ofD′.

In some embodiments, R″₇ is a direct or indirect linkage to L^(C), R₇ or

in which

denotes direct or indirect linkage to L^(C) via a functional group ofR₇.

In some embodiments, R″₇ is a direct or indirect linkage to L^(D), R₇ or

in which

denotes direct or indirect linkage to L^(D) via a functional group ofR₇.

In some embodiments, R″₁₀ is a direct or indirect linkage to L^(C), R₁₀,or

in which

denotes direct or indirect linkage L^(C) via a functional group of R₁₀.

In some embodiments, R″₁₀ is a direct or indirect linkage to L^(D), R₁₀,or

in which

denotes direct or indirect linkage L^(C) via a functional group of R₁₀.

In some embodiments, E″ is a direct or indirect linkage to the PBRM; D″is D′; R″₇ is R and R″₁₀ is R₁₀.

In some embodiments, E″ is a direct or indirect linkage to L^(C); D″ isD′; R″₇ is R₇ and R″₁₀ is R₁₀.

In some embodiments, E″ is a direct or indirect linkage to L^(D); D″ isD′; R″₇ is R₇ and R″₁₀ is R₁₀.

In some embodiments, E″ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofE; D″ is D′; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, E″ is

in which

denotes direct or indirect linkage to L^(C) via a functional group of E;D″ is D′; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, E″ is

in which

denotes direct or indirect linkage to L^(D) via a functional group of E;D″ is D′; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, D″ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofD; E″ is E; R″₇ is R₇ and R″₁₀ is R₁₀.

In some embodiments, D″ is

in which

denotes direct or indirect linkage to L^(C) via a functional group of D;E″ is E; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, D″ is

in which

denotes direct or indirect linkage to L^(D) via a functional group of D;E″ is E; R″₇ is R₇; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is a direct or indirect linkage to the PBRM; E″is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is a direct or indirect linkage to L^(C); E″ isE; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is a direct or indirect linkage to L^(D); E″ isE; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofR₇; E″ is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is

in which

denotes direct or indirect linkage to L^(C) via a functional group ofR₇; E″ is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₇ is

in which

denotes direct or indirect linkage to L^(D) via a functional group ofR₇, E″ is E; D″ is D′; and R″₁₀ is R₁₀.

In some embodiments, R″₁₀ is a direct or indirect linkage to the PBRM;E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is a direct or indirect linkage to L^(C); E″is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is a direct or indirect linkage to L^(D); E″is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is

in which

denotes direct or indirect linkage to the PBRM via a functional group ofR₁₀; E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is

in which

denotes direct or indirect linkage to L^(C) via a functional group ofR₁₀; E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, R″₁₀ is

in which

denotes direct or indirect linkage to L^(D) via a functional group ofR₁₀; E″ is E; D″ is D′; and R″₇ is R₇.

In some embodiments, the conjugates of Formula (IV) include those whereeach of the moieties defined for one of E″, D″, R″₇, R″₁₀, D′, T, E, A,R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆,R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₃₁, R₃₂, R₃₃, R₃₄, R_(35a), R_(35b), R_(36a),R_(36b), R_(36c), R_(36d), R_(37a), R_(37b), R_(a), R^(b), R^(N), R^(Q),X₀, Y₀, Z₀, X₁, Y₁, Z₁, X₂, X₃, M, Q, m, n, r, s, t, and x, can becombined with any of the moieties defined for the others of E″, D″, R″₇,R″₁₀, D′, T, E, A, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, R₁₅, R₁₆, R₁₇R₁₈, R₁₉, R₂₀, R₂₁, R₃₁, R₃₂, R₃₃, R₃₄, R_(35a),R_(35b), R_(36a), R_(36c), R_(36d), R_(37a), R_(37b), R₄₀, R_(a), R^(b),R_(N), R^(Q), X₀, Y₀, Z₀, X₁, Y₁, Z₁, X₂, X₃, X₄, X₈, M, Q, m, n, r, s,t, and x.

In some embodiments, D′ is D1, D2, D3, or D4:

wherein the dotted line between C2 and C3 or between C2 and C1 in D1 orthe dotted line in D4 indicates the presence of a single or double bond;and

m is 0, 1 or 2;

when D′ is D1, the dotted line between C2 and C3 is a double bond, and mis 1, then R₁ is:

(i) C₆₋₁₀ aryl group, optionally substituted by one or more substituentsselected from —OH, halo, —NO₂, —CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂,—OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, a polyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a), 3- to14-membered heterocycloalkyl, 5- to 12-membered heteroaryl, bis-oxy-C₁₋₃alkylene, —NR₃R₄, —S(═O)₂R₁₂, —S(═O)₂NR₁₃R₁₄, —SR₁₂, —SO_(x)M,—OSO_(x)M, —NR₉COR₁₉, —NH(C═NH)NH₂;

(ii) C₁₋₅ alkyl;

(iii) C₃₋₆ cycloalkyl;

(viii) halo;

when D′ is D1, the dotted line between C2 and C3 is a single bond, and mis 1, then R₁ is:

(i) —OH, ═O, ═CH₂, —CN, —R₂, —OR₂, halo, ═CH—R₆, ═C(R₆)₂, —O—SO₂R₂,—CO₂R₂, —COR₂, —CHO, or —COOH; or

when D′ is D1 and m is 2, then each R₁ independently is halo and eitherboth R₁ are attached to the same carbon atom or one is attached to C₂and the other is attached to C₃;

T is C₁₋₁₀ alkylene linker;

A is

wherein the —NH group of A is connected to the —C(O)-T- moiety ofFormula (I) and the C═O moiety of A is connected to E; and each

independently is

E is E1, E2, E3, E4, —OH, —NH—(C₁₋₆ alkylene)-R_(13a), —O—(CH₂)₃—NH₂,—O—CH(CH₃)—(CH₂)₂—NH₂ or —NH—(CH₂)₃—O—C(═O)—CH(CH₃)—NH₂:

wherein the dotted line in E1 or E4 indicates the presence of a singleor double bond;

each occurrence of R₂ and R₃ independently is an optionally substitutedC₁₋₈ alkyl, optionally substituted C₂₋₈ alkenyl, optionally substitutedC₂₋₈ alkynyl, optionally substituted C₃₋₈ cycloalkyl, optionallysubstituted 3- to 20-membered heterocycloalkyl, optionally substitutedC₆₋₂₀ aryl or optionally substituted 5- to 20-membered heteroaryl, and,optionally in relation to the group NR₂R₃, R₂ and R₃ together with thenitrogen atom to which they are attached form an optionally substituted4-, 5-, 6- or 7-membered heterocycloalkyl or an optionally substituted5- or 6-membered heteroaryl;

R₄, R₅ and R₇ are each independently —H, —R₂, —OH, —OR₂, —SH, —SR₂,—NH₂, —NHR₂, —NR₂R₃, —NO₂, —SnMe₃, halo or a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a); or R₄ and R₇ together form bis-oxy-C₁₋₃ alkylene;

each R₆ independently is —H, —R₂, —CO₂R₂, —COR₂, —CHO, —CO₂H, or halo;

each R₈ independently is —OH, halo, —NO₂, —CN, —N₃, —OR₂, —COOH, —COOR₂,—COR₂, —OCONR₁₃R₁₄, —CONR₁₃R₁₄, —CO—NH—(C₁₋₆ alkylene)-R_(13a), C₁₋₁₀alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethyleneglycol unit —(OCH₂CH₂)_(r)—OR_(a), 3- to 14-membered heterocycloalkyl,5- to 12-membered heteroaryl, —S(═O)₂R₁₂, —S(═O)₂NR₁₃R₁₄, —SR₁₂,—SO_(x)M, —OSO_(x)M, —NR₉COR₁₉, —NH(C═NH)NH₂, —R₂₀—R₂₁—NR₃R₄,—R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃;

each R₉ independently is C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl;

R¹⁰ is —H or a nitrogen protecting group;

R¹¹ is -QR^(Q) or —SO_(x)M;

or R¹⁰ and R¹¹ taken together with the nitrogen atom and carbon atom towhich they are respectively attached, form a N═C double bond;

each R¹² independently is C₁₋₇ alkyl, 3- to 20-memberedheterocycloalkyl, 5- to 20-membered heteroaryl, or C₆₋₂₀ aryl;

each occurrence of R₁₃ and R₁₄ are each independently H, C₁₋₁₀ alkyl, 3-to 20-membered heterocycloalkyl, 5- to 20-membered heteroaryl, or C₆₋₂₀aryl;

each R_(13a) independently is —OH or —NR₁₃R₁₄,

R₁₅, R₁₆, R₁₇ and R₁₈ are each independently —H, —OH, halo, —NO₂, —CN,—N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3-14 membered heterocycloalkyl, 5- to 12-memberedheteroaryl, —NR₁₃R₁₄, —S(═O)₂R₂, —S(═O)₂NR₁₃R₁₄, —SR₂, —SO_(x)M,—OSO_(x)M, —NR₉COR₁₉ or —NH(C═NH)NH₂;

each R₁₉ independently is C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenylor C₂₋₁₀ alkynyl;

each R₂₀ independently is a bond, C₆₋₁₀ arylene, 3-14 memberedheterocycloalkylene or 5- to 12-membered heteroarylene;

each R₂₁ independently is a bond or C₁₋₁₀ alkylene;

R₃₁, R₃₂ and R₃₃ are each independently —H, C₁₋₃ alkyl, C₂₋₃ alkenyl,C₂₋₃ alkynyl or cyclopropyl, wherein the total number of carbon atoms inthe R₁ group is no more than 5;

R₃₄ is —H, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, cyclopropyl, orphenyl wherein the phenyl is optionally substituted by one or more ofhalo, methyl, methoxy, pyridyl or thiophenyl;

one of R_(35a) and R_(35b) is —H and the other is a phenyl groupoptionally substituted with one or more of halo, methyl, methoxy,pyridyl or thiophenyl;

R_(36a), R_(36b), R_(36c) are each independently —H or C₁₋₂ alkyl;

R_(36d) is —OH, —SH, —COOH, —C(O)H, —N═C═, —NHNH₂, —CONHNH₂,

or NHR^(N), wherein R^(N) is —H or C₁₋₄ alkyl;

R_(37a) and R_(37b) are each independently is —H, —F, C₁₋₄ alkyl, C₂₋₃alkenyl, wherein the alkyl and alkenyl groups are optionally substitutedby C₁₋₄ alkyl amido or C₁₋₄ alkyl ester; or when one of R_(37a) andR_(37b) is —H, the other is —CN or a C₁₋₄ alkyl ester;

R₃₈ and R₃₉ are each independently H, R₁₃, ═CH₂, ═CH—(CH₂)_(s1)—CH₃, ═O,(CH₂)_(s1)—OR₁₃, (CH₂)_(s1)—CO₂R₁₃(CH₂)_(s1)—NR₁₃R₁₄, O—(CH₂)₂—NR₁₃R₁₄,NH—C(O)—R₁₃, O—(CH₂)s-NH—C(O)—R₁₃, O—(CH₂)s-C(O)NHR₃,(CH₂)_(s1)OS(═O)₂R₃, O—SO₂R₃, (CH₂)_(s1)—C(O)R₁ and(CH₂)_(s1)—C(O)NR₁₃R₁₄;

X₀ is CH₂, NR₆, C═O, BH, SO or SO₂;

Y₀ is O, CH₂, NR₆ or S;

Z₀ is absent or (CH₂)_(n);

each X₁ independently is CR_(b), or N;

each Y₁ independently is CH, NR_(a), O or S;

each Z₁ independently is CH, NR_(a), O or S;

each R_(a) independently is H or C₁₋₄ alkyl;

each R_(b) independently is H, OH, C₁₋₄ alkyl, or C₁₋₄ alkoxyl;

X₂ is CH, CH₂ or N;

X₃ is CH or N;

X₄ is NH, O or S;

X₈ is NH, O or S;

Q is O, S or NH;

when Q is S or NH, then R^(Q) is —H or optionally substituted C₁₋₂alkyl; or

when Q is O, then R^(Q) is —H or optionally substituted C₁₋₂ alkyl,—SO_(x)M, —PO₃M, —(CH₂—CH₂—O)_(n9)CH₃, —(CH₂—CH₂O)_(n9)—CH₂)₂—R₄₀,—C(O)—(CH₂—CH₂—O)_(n9)CH₃, —C(O)O—(CH₂—CH₂—O)_(n9)CH₃.—C(O)NH—(CH₂—CH₂—O)_(n9)CH₃,—(CH₂)_(n)—NH—C(O)—CH₂—O—CH₂—C(O)—NH—(CH₂—CH₂—O)₉CH₃,—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)—(CH₂—CH₂—O)_(n9)CH₃, a sugar moiety,

each M independently is H or a monovalent pharmaceutically acceptablecation;

n is 1, 2 or 3;

each r independently is an integer from 1 to 200;

s is 1, 2, 3, 4, 5 or 6;

s₁ is 0, 1, 2, 3, 4, 5 or 6:

n₉ is 1, 2, 3, 4, 5, 6, 8, 12 or 24;

t is 0, 1, or 2;

R₄₀ is —SO₃H, —COOH, —C(O)NH(CH₂)₂SO₃H or —C(O)NH(CH₂)₂COOH; and

each x independently is 2 or 3.

The PBD drug moiety of Formula (IV) can have one or more of thefollowing features when applicable:

In some embodiments, the PBD drug moiety of Formula (IV) is of Formula(IV-a),

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of Formulae (IV-a) include thosewhere each of the moieties defined for one of E″, A, R₄, R₅, R″₇, R″₁₀,R₁₁, and D″ can be combined with any of the moieties defined for theothers of E″, A, R₄, R₅, R″₇, R″₁₀, R₁₁, and D″.

In some embodiments, D′ is D1.

In some embodiments, D′ is D2.

In some embodiments, D′ is D3.

In some embodiments, D′ is D4

In some embodiments, the PBD drug moiety of Formula (IV) is of any oneof formulae (V-1), (V-2), and (V-3):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of any one of Formulae (V-1)-(V-3)include those where each of the moieties defined for one of E″, A, R₁,R₄, R₅, R″₇, R″₁₀, R₁₁, and m can be combined with any of the moietiesdefined for the others of E″, A, R₁, R₄, R₅, R″₇, R″₁₀, R₁₁, and m.

In some embodiments, the PBD drug moiety of Formula (IV) is of Formula(VI-1):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of Formula (VI-1) include thosewhere each of the moieties defined for one of E″, A, R₄, R₅, R″₇, R″₁₀,R₁₁, R₁₅, R₁₆, R₁₇, and R₁₈ can be combined with any of the moietiesdefined for the others of E″, A, R₄, R₅, R″₇, R″₁₀, R₁₅, R₁₆, R₇, andR₁₈.

In some embodiments, the PBD drug moiety of Formula (IV) is of Formula(VII), (VII-1), (VII-2), or (VII-3):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of any one of Formulae (VII),(VII-1), (VII-2), and (VII-3) include those where each of the moietiesdefined for one of E″, A, R₄, R₅, R″₇, R″₁₀, R₁₁, R₃₈, and R₃₉, whereapplicable, can be combined with any of the moieties defined for theothers of E″, A, R₄, R₅, R″₇, R″₁₀, R₁₁, R₃₈, and R₃₉.

In some embodiments, the PBD drug moiety of Formula (IV) is of Formula(VIII):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of Formula (VIII) include thosewhere each of the moieties defined for one of E″, A, R₄, R₅, R″₇, R″₁₀,R₁₁, X₀, and Y₀ can be combined with any of the moieties defined for theothers of E″, A, R₄, R₅, R″₇, R″₁₀, R₁₁, X₀, and Y₀.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, optionallysubstituted by one or more substituents selected from —OH, halo, —NO₂,—CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3- to 14-membered heterocycloalkyl, 5- to12-membered heteroaryl, bis-oxy-C₁₋₃ alkylene, —NR₁₃R₁₄, —S(═O)₂R₁₂,—S(═O)₂NR₁₃R₁₄, —SR₁₂, —SO_(x)M, —OSO_(x)M, —NR₉COR₁₉, and —NH(C═NH)NH₂.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, optionallysubstituted by one or more substituents selected from —OH, halo, —NO₂,—CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄. C₁₋₁₀ alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3- to 14-membered heterocycloalkyl, 5- to12-membered heteroaryl, —NR₁₃R₁₄, —S(═O)₂R₂, —S(═O)₂NR₁₃R₁₄, —SR₁₂,—NR₉COR₁₉, and —NH(C═NH)NH₂.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, optionallysubstituted by one or more substituents selected from —OH, halo, —OR₂,—COOH, —COOR₂, —COR₂, 3- to 14-membered heterocycloalkyl, and —NR₁₃R₁₄.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone or more substituents selected from —OH, halo, —OR₂, —COH, —COOR₂,—COR₂, 3- to 14-membered heterocycloalkyl, and —NR₁₃R₁₄.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone substituent selected from —OH, halo, —OR₂, —COOH, —COOR₂, —COR₂, 3-to 14-membered heterocycloalkyl, and —NR₁₃R₁₄.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone substituent selected from —OH, —OR₂, —COOH, —COOR₂, 3- to14-membered heterocycloalkyl, and —NR₁₃R₁₄.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone substituent selected from —OH, and —COOH.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone substituent selected from —OR₂— and —COOR₂.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone 3- to 14-membered heterocycloalkyl.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₆₋₁₀ aryl group, substituted byone —NR₁₃R₁₄.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₁₋₅ alkyl.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is C₃₋₆ cycloalkyl.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is cyclopropyl.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is R

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa double bond, and m is 1, then R₁ is halo.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa single bond, and m is 1, then R₁ is: —OH, ═O, ═CH₂, —CN, —R₂, —OR₂,halo, ═CH—R₆, ═C(R₆)₂, —O—SO₂R₂, —CO₂R₂, —COR₂, —CHO, or —COOH.

In some embodiments, when D′ is D1, the dotted line between C₂ and C₃ isa single bond, and m is 1, then R₁ is: ═CH₂, ═CH—R₆ or ═C(R₆)₂.

In some embodiments, when D′ is D1 and m is 2, then each R₁independently is halo and either both R₁ are attached to the same carbonatom or one is attached to C₂ and the other is attached to C₃.

In some embodiments, when D′ is D4, the dotted line is a single bond,and R₃₈ and R₃₉ are each hydrogen.

In some embodiments, T is C₂₋₆ alkylene linker.

In some embodiments, T is C₂₋₄ alkylene linker.

In some embodiments, T is butylene.

In some embodiments, T is propylene

In some embodiments, T is n-propylene.

In some embodiments, T is ethylene.

In some embodiments, each

independently is

In some embodiments, each

independently is

In some embodiments, s is 1, 2, 3, 4 or 5.

In some embodiments, s is 2, 3, 4, 5 or 6.

In some embodiments, s is 1, 2, 3 or 4.

In some embodiments, s is 2, 3, 4 or 5.

In some embodiments, s is 3, 4, 5 or 6.

In some embodiments, s is 1, 2 or 3.

In some embodiments, s is 2, 3 or 4.

In some embodiments, s is 3, 4 or 5.

In some embodiments, s is 4, 5 or 6.

In some embodiments, s is 1 or 2.

In some embodiments, s is 2 or 3.

In some embodiments, s is 3 or 4.

In some embodiments, s is 4 or 5.

In some embodiments, s is 5 or 6.

In some embodiments, s is 1.

In some embodiments, s is 2.

In some embodiments, s is 3.

In some embodiments, s is 4.

In some embodiments, s is 5.

In some embodiments, s is 6.

In some embodiments, A is

In some embodiments, A is

wherein each X₁ independently is CH or N.

In some embodiments, A is

In some embodiments, A is

wherein each X₁ independently is CH or N.

In some embodiments, A is

In some embodiments, A is:

wherein each X₁ independently is CH or N.

In some embodiments, A is:

In some embodiments, t is 0.

In some embodiments, t is 1.

In some embodiments, t is 2.

In some embodiments E is -E1, E2, E3 E4, OH, —NH—(C₁₋₆alkylene)-R_(13a), —O—(CH₂)₃—NH₂, —O—CH(CH₃)—(CH₂)₂—NH₂ or—NH—(CH₂)₃—O—C(═O)—CH(CH₃)—NH₂

wherein the dotted line in E1 or E4 indicates the presence of a singleor double bond.

In some embodiments, E is

In some embodiments, E is

In some embodiments, X₃ is CH. In some embodiments, X₃ is N.

In some embodiments, when E is

then s is 2, 3, 4, 5 or 6, In some embodiments, s is 2, s is 3, s is 4,s is 5 or s is 6.

In some embodiments, when X₄ is O or S, then s is 2, 3, 4, 5 or 6, Insome embodiments, s is 2, s is 3, s is 4, s is 5 or s is 6.

In some embodiments, E is

In some embodiments, E is —OH.

In some embodiments, E is —NH—(C₁₋₆ alkylene)-OH, wherein C₁₋₆ alkyleneis a linear or branched alkylene.

In some embodiments, E is —NH—(CH₂)_(u)—OH, in which u is 1, 2, 3, 4, 5,or 6.

In some embodiments, E is —NH—(CH₂)₃—OH.

In some embodiments, E is —O—(CH₂)₃—NH₂.

In some embodiments, E is —O—CH(CH₃)—(CH₂)₂—NH₂,

In some embodiments, E is —NH—(CH₂)₃—O—C(═O)—CH(CH₃)—NH₂

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is N

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

In some embodiments, E is

is the functional group of E.

In some embodiments, in

the

denotes direct or indirect linkage to the PBRM.

In some embodiments, in

the

denotes direct or indirect linkage to L^(C).

In some embodiments, in

the

denotes direct or indirect linkage to L^(D).

In some embodiments, in

the functional group of E is R₈ or a portion thereof.

In some embodiments, in

the

denotes direct or indirect linkage to the PBRM via R₈ or a portionthereof.

In some embodiments, in

the

denotes direct or indirect linkage to L^(C) via R₈ or a portion thereof.

In some embodiments, in

the

denotes direct or indirect linkage to L^(D) via R⁸ or a portion thereof.

In some embodiments, each R₈ independently is —OH, halo, —NO₂, —CN, —N₃,—OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, —CO—NH—(C₁₋₆ alkylene)-R_(13a),—OCO—NH—(C₁₋₆ alkylene)-R_(13a), C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, a polyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a), 3- to14-membered heterocycloalkyl, 5- to 12-membered heteroaryl, —S(═O)₂R₁₂,—S(═O)₂NR₁₃R₁₄, —SO_(x)M, —OSO_(x)M, —NR₉COR₁₉, —R₂₀—R₂₁—NR₁₃R₁₄,—R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃

In some embodiments, each R₈ independently is —CONR₁₃R₁₄.

In some embodiments, when E is

then at least one R₈ is —CONR₁₃R₁₄.

In some embodiments, when E is

then at least one R₈ is —R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃.

In some embodiments, when E is

then at least one R₈ is —O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃.

In some embodiments, each R₈ independently is —CO—NH—(C₁₋₆alkylene)-R_(13a) or —OCO—NH—(C₁₋₆ alkylene)-R_(13a).

In some embodiments, when E is

then at least one R₈ is —CO—NH—(C₁₋₆ alkylene)-R_(13a) or —OCO—NH—(C₁₋₆alkylene)-R_(13a).

In some embodiments, each R₈ independently is —OH, halo, —NO₂, —CN, —N₃,—OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3- to 14-membered heterocycloalkyl, 5- to12-membered heteroaryl, —S(═O)₂R₁₂, —S(═O)₂NR₁₃R₁₄, —SR₂, —SO_(x)M,—OSO_(x)M, —NR₉COR₁₉, —NH(C═NH)NH₂, —R₂₀—R₂₁—NR₁₃OR₁₄,—R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃.

In some embodiments, each R₈ independently is —OH, —OR₂, —COOH, —COOR₂,—COR₂, —OCONR₁₃R₄, —CONR₁₃R₁₄, —CO—NH—(C₁₋₆ alkylene)-R_(13a), apolyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a), 3- to 7-memberedheterocycloalkyl, 5- to 6-membered heteroaryl, —S(═O)₂R₂,—S(═O)₂NR₁₃R₁₄, —SR₂, —R₂₀—R₂₁—NR₁₃R₁₄,—R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃

wherein R₁₃ and R₁₄ are each independently —H or C₁₋₁₀ alkyl;

each R₂₀ is phenylene; and

each R₂₁ independently is C₁₋₄ alkylene.

In some embodiments, each R₈ independently is —OH, —OR₂, —COOH, —COOR₂,—COR₂, —S(═O)₂R₁₂, —SR₁₂, —R₂₀—R₂₁—NR₁₃R₁₄,—R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃.

In some embodiments, each R₈ independently is —OH or —OR₂.

In some embodiments, each R₈ independently is —COH, —COOR₂, or —COR₂.

In some embodiments, each R₈ independently is —S(═O)₂R₂ or —SR₂.

In some embodiments, each R₈ independently is —CONR₁₃R₁₄ or —CO—NH—(C₁₋₆alkylene)-R_(13a).

In some embodiments, each R₈ independently is —R₂₀—R₂₁—NR₃R₄.

In some embodiments, R₈ is —NH₂.

In some embodiments, R₈ is —CH₂NH₂.

In some embodiments, R₈ is —CH₂CH₂NH₂.

In some embodiments, R₈ is —CH₂CH₂CH₂NH₂.

In some embodiments, R₈ is —NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃.

In some embodiments, R₈ is —NH—P(O)(OH)—(OCH₂CH₂)—OCH₃.

In some embodiments, R₈ is —O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃.

In some embodiments, R₈ is —O—P(O)(OH)—(OCH₂CH₂)—OCH₃.

In some embodiments, each R_(13a) independently is OH or NHR₃.

In some embodiments, each occurrence of R₁₃ is independently H or C₁₋₁₀alkyl (e.g., C₁₋₆ alkyl).

In some embodiments, each occurrence of R₁₄ is independently H or C₁₋₁₀alkyl (e.g., C₁₋₆ alkyl).

In some embodiments, each occurrence of R₁₃ is independently 3- to20-membered (e.g., 4- to 14-membered) heterocycloalkyl or 5- to20-membered (e.g., 5- to 10-membered) heteroaryl.

In some embodiments, each occurrence of R₁₄ is independently 3- to20-membered (e.g., 4- to 14-membered) heterocycloalkyl or 5- to20-membered (e.g., 5- to 10-membered) heteroaryl.

In some embodiments, R₄, R₅ and R₇ are each independently —H, —R₂, —OH,—OR₂, —SH, —SR₂, —NH₂, —NHR₂, —NR₂R₃, —NO₂, halo or a polyethyleneglycol unit —(OCH₂CH₂)_(r)—OR_(a).

In some embodiments, at least one of R₄, R₅ and R₇ is —OR₂.

In some embodiments, at least one of R₄, R₅ and R₇ is a polyethyleneglycol unit —(OCH₂CH₂)_(r)—OR_(a).

In some embodiments, at least two of R₄, R₅ and R₇ are —H.

In some embodiments, two of R₄, R₅ and R₇ are —H, and the other is —OR₂.

In some embodiments, two of R₄, R₅ and R₇ are —H, and the other is—OCH₃.

In some embodiments, R₄ and R₅ are each —H, and R₇ is —OCH₃.

In some embodiments, R₄ and R₅ are each —H, and R₇ is—(OCH₂CH₂)_(r)—OR_(a).

In some embodiments, R₄ and R₇ together form bis-oxy-C₁₋₃ alkylene.

In some embodiments, each of R₂₀ and R₂₁ is a bond.

In some embodiments, one of R₂₀ and R₂₁ is a bond and the other is not abond.

In some embodiments, R₂₀ is a bond and R₂₁ is not a bond.

In some embodiments, R₂₀ is a bond and R₂₁ is C₁₋₁₀ alkylene.

In some embodiments, R₂₁ is a bond and R₂₀ is not a bond.

In some embodiments, R₂₁ is a bond and R₂₀ is C₆₋₁₀ arylene, 3-14membered heterocycloalkylene or 5- to 12-membered heteroarylene.

In some embodiments, R¹⁰ and R¹¹ taken together with the nitrogen atomand carbon atom to which they are respectively attached, form a N═Cdouble bond.

In some embodiments, R¹⁰ is —H or a nitrogen protecting group, and R″ is-QR^(Q).

In some embodiments, R¹⁰ is —H and R¹¹ is -QR^(Q).

In some embodiments, R¹⁰ is a nitrogen protecting group and R″ is-QR^(Q), wherein the nitrogen protecting group is allyloxycarbonyl(alloc), carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz or MeOZ),acetyl (Ac), benzoyl (Bz), benzyl (Bn), trichloroethoxycarbonyl (Troc),t-butoxycarbonyl (BOC) or 9-fluorenylmethylenoxycarbonyl (Fmoc).

In some embodiments, R¹¹ is —OSO_(x)M.

In some embodiments, R¹¹ is —SO_(x)M.

In some embodiments, R¹¹ is —OH.

In some embodiments, R¹¹ is —OPO₃M.

In some embodiments, R¹¹ is —O(CH₂CH₂O)_(n9)CH₃.

In some embodiments, R¹¹ is —O—(CH₂—CH₂O)_(n9)—(CH₂)₂—R₄₀.

In some embodiments, R¹¹ is —OC(O)O—(CH₂—CH₂—O)_(n9)CH₃.

In some embodiments, R¹¹ is —OC(O)NH—(CH₂—CH₂—O)_(n9)CH₃.

In some embodiments, R¹¹is-O—(CH₂)_(n)—NH—C(O)—CH₂—O—CH₂—C(O)—NH—(CH₂—CH₂—O)_(n9)CH₃.

In some embodiments, R¹¹ is —O—(CH₂),—NH—C(O)—(CH₂)—(CH₂—CH₂—O)_(n9)CH₃,

In some embodiments, R¹¹ is —O-sugar moiety.

In some embodiments, R¹⁵, R₁₆, R₁₇ and R₁₈ are each independently —H,—OH, halo, —NO₂, —CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄,C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, apolyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a), 3-14 memberedheterocycloalkyl, 5- to 12-membered heteroaryl, —NR₁₃R₁₄, —S(═O)₂R₁₂,—S(═O)₂NR₁₃R₁₄, —SR₁₂ or —NH(C═NH)NH₂.

In some embodiments, at least one of R₁₅, R₁₆, R₁₇ and R₁₈ is —H.

In some embodiments, at least two of R₁₅, R₁₆, R₁₇ and R₁₈ is —H.

In some embodiments, at least three of R₁₅, R₁₆, R₁₇ and R₁₈ is —H.

In some embodiments, R₁₅, R₁₆, R₁₇ and R₁₈ are each —H or —NR₁₃R₁₄.

In some embodiments, at least one of R₁₅, R₁₆, R₁₇ and R₁₈ is —NR₁₃R₁₄.

In some embodiments, at least one of R₁₅, R₁₆, R₁₇ and R₁₈ is —NH₂.

In some embodiments, one of R₁₅, R₁₆, R₁₇ and R₁₈ is —NR₃R₄.

In some embodiments, one of R₁₅, R₁₆, R₁₇ and R₁₈ is —NH₂.

In some embodiments, R₁₆, R₁₇ and R₁₈ are each —H; and R₁₅ is —NH₂.

In some embodiments, R₁₅, R₁₇ and R₁₈ are each —H; and R₁₆ is —NH₂.

In some embodiments, R₁₅, R₁₆ and R₁₈ are each —H; and R₁₇ is —NH₂.

In some embodiments, R₁₅, R₁₆ and R₁₇ are each —H; and Rig is —NH₂.

In some embodiments, X₀ is CH₂, NR₆, or C═O.

In some embodiments, Y₀ is O, CH₂, or NR₆.

In some embodiments, Z₀ is absent.

In some embodiments, Z₀ is (CH₂)_(n), and n is 1 or 2.

In some embodiments, when Q is S or NH, then R^(Q) is —H.

In some embodiments, when Q is S or NH, then R^(Q) is optionallysubstituted C₁₋₂ alkyl.

In some embodiments, when Q is O, then R^(Q) is —H.

In some embodiments, when Q is O, then R^(Q) is optionally substitutedC₁₋₂ alkyl.

In some embodiments, when Q is O, then R^(Q) is —SO_(x)M.

In some embodiments, when Q is O, then R^(Q) is hydrogen.

In some embodiments, when Q is O, then R^(Q) is —PO₃M.

In some embodiments, when Q is O, then R^(Q) is —(CH₂—CH₂—O)_(n9)CH₃,and n is 6, 8, 12 or 24.

In some embodiments, when Q is O, then R^(Q) is—C(O)—(CH₂—CH₂—O)_(n9)CH₃ and n is 6, 8, 12 or 24

In some embodiments, when Q is O, then R^(Q) is —C(O)—(CH₂—CH₂—O)₉CH₃and n₉ is 6, 8, 12 or 24.

In some embodiments, when Q is O, then R^(Q) is—C(O)NH—(CH₂—CH₂—O)_(n9)CH₃ and n₉ is 6, 8, 12 or 24.

In some embodiments, when Q is O, then R^(Q) is—(CH₂)—NH—C(O)—CH₂—O—CH₂—C(O)—NH—(CH₂—CH₂—O)_(n9)CH₃, and n is 2 and n₉is 6, 8, 12 or 24.

In some embodiments, when Q is O, then R^(Q) is—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)—(CH₂—CH₂—O)_(n9)CH₃, and n is 2 and n₉ is6, 8, 12 or 24.

In some embodiments, when Q is O, then R^(Q) is a -sugar moiety.

In some embodiments, when Q is O, then R^(Q) is

In some embodiments, when Q is O, then R^(Q) is

In some embodiments, when Q is O, then R^(Q) is

In some embodiments, when Q is O, then R^(Q) is

In some embodiments, when Q is O, then R^(Q) is

In some embodiments, when Q is O, then R^(Q) is.

and n₉ is 6, 8, 12 or 24

In some embodiments, when Q is O, then R^(Q)

and n₉ is 6, 8, 12 or 24

In some embodiments, the compound of Formula (I) contains at most one—SO_(x)M or —OSO_(x)M.

In some embodiments, R¹¹ is —OSO_(x)M, —SO_(x)M, —OH, —OCH₃,O—(CH₂)₂—NH—C(O)—CH₂—O—CH₂—C(O)—NH—(CH₂—CH₂—O)₈CH₃.

In some embodiments,

is

in which

denotes a direct or indirect linkage to the PBRM, L^(C), or L^(D), and

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).

In some embodiments,

is

in which

denotes a direct or indirect linkage to the PBRM, L^(C), or L^(D), and

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).

In some embodiments,

is

in which

denotes a direct or indirect linkage to the PBRM, L^(C), or L^(D), and

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).

In some embodiments,

is

in which

denotes a direct or indirect linkage to the PBRM, L^(C), or L^(D), and

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).

In some embodiments, E is

in which

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).

In some embodiments, E is

in which

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).

In some embodiments, the PBD drug moiety of Formula (IV) is of any oneof Formulae (IX-a to IX-r):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of any one of Formulae (IX-a)-(IX-r)include those where each of the moieties defined for one of E″, A, R₄,R₅, R″₇, R″₁₀, and R₁ can be combined with any of the moieties definedfor the others of E″, A, R₄, R₅, R″₇, R″₁₀, and R₁₁.

In some embodiments, the PBD drug moiety of Formula (IV) is of any oneof Formulae (X-a) to (X-c):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of any one of Formulae (X-a)-(X-c)include those where each of the moieties defined for one of E″, A, R₄,R₅, R″₇, R″₁₀, and R₁₁ can be combined with any of the moieties definedfor the others of E″, A, R₄, R₅, R″₇, R″₁₀, and R₁₁.

In some embodiments, the PBD drug moiety of Formula (IV) is of any oneof Formulae (XI-a) to (XI-c):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of any one of Formulae (XI-a)-(XI-c)include those where each of the moieties defined for one of E″, A, R₄,R₅, R″₇, R″₁₀, and R₁₁ can be combined with any of the moieties definedfor the others of E″, A, R₄, R₅, R″₇, R″₁₀, and R₁₁.

In some embodiments, the PBD drug moiety of Formula (IV) is

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer, wherein:

R₁₃ is H;

p is 1, 2, 3 or 4, and

E″, R″₇, R″₁₀ and R₁₁ are as defined herein.

In some embodiments, the PBD drug moiety of Formula (IV) is of Formula(XII):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of Formula (XII) include those whereeach of the moieties defined for one of E″, A, T, R₄, R₅, R″₇, R″₁₀,R₁₁, X₄, and D″ can be combined with any of the moieties defined for theothers of E″, A, T, R₄, R₅, R″₇, R″₁₀, R₁₁, X₄, and D″.

In the PBD drug moiety of Formula (XII) above, X₄ is C═S, CH₂, SO, SO₂or BH; and E″, A, T, D″, R₄, R₅, R″₇, R″₁₀ and R₁₁ are as definedherein.

In some embodiments, the PBD drug moiety of Formula (XII) is of any oneof Formulae (XII-a) to (XII-e):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the conjugates of any one of Formulae (XIIa)-(XIIe)include those where each of the moieties defined for one of E″, A, T,R₄, R₅, R″₇, R″₁₀, R₁₁, and D″ can be combined with any of the moietiesdefined for the others of E″, A, T, R₄, R₅, R″₇, R″₁₀, R₁₁, and D″.

In some embodiments, the PBD drug moiety (D), prior to being connectedto another portion of the conjugate (e.g., the linker unit (L^(C))),corresponds to a compound selected from the compounds listed in Table 1,tautomers thereof, pharmaceutically acceptable salts or solvatesthereof, or pharmaceutically acceptable salts or solvates of thetautomers.

TABLE I Structure

In some embodiments, the PBD drug moiety (D), prior to being connectedto another portion of the conjugate (e.g., the linker unit (L^(C))),corresponds to a compound of any one of Formula (XIIIa) to (XIIIm):

a tautomer thereof, a pharmaceutically acceptable salt or solvatethereof, or a pharmaceutically acceptable salt or solvate of thetautomer.

In some embodiments, the PBD drug moiety (D), connected to anotherportion of the conjugate (e.g., the linker unit (L^(C))), corresponds toa conjugate selected from the conjugates listed in Table 1A, tautomersthereof, pharmaceutically acceptable salts or solvates thereof, orpharmaceutically acceptable salts or solvates of the tautomers, wherein

indicates the point of attachment to the linker unit.

TABLE 1A

Representative examples of conjugates of Formula (I) include thoselisted in Table 2, a tautomer thereof, a pharmaceutically acceptablesalt or solvate thereof, or a pharmaceutically acceptable salt orsolvate of the tautomer.

TABLE 2 Conjugate No. Structure Conjugate No. 5 (Example 1)

Conjugate No. 8 (Example 2)

Conjugate No. 14 (Example 4)

Conjugate No. 22 (Example 5)

Conjugate No. 28 (Example 6)

Conjugate No. 34 (Example 7)

Conjugate No. 35 (Example 8)

Conjugate No. 101

Conjugate No. 102

Conjugate No. 103

Conjugate No. 104

Conjugate No. 105

Conjugate No. 106

Conjugate No. 107

Conjugate No. 108

Conjugate No. 109

Conjugate No. 110

Conjugate No. 111

Conjugate No. 112

Conjugate No. 113

Conjugate No. 114

Conjugate No. 115

Conjugate No. 116

Conjugate No. 118

wherein n₈=6, 8 or 12 and, preferably, d₁₃ is 3 to 5.

In some embodiments, the PBD conjugates is a conjugate of any one ofFormulae (XIVa) to (XIVx):

pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVa), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVa).

In some embodiments, the PBD conjugate is of Formula (XIVb), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVb).

In some embodiments, the PBD conjugate is of Formula (XIVc), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVc).

In some embodiments, the PBD conjugate is of Formula (XIVd), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVd).

In some embodiments, the PBD conjugate is of Formula (XIVe), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVe).

In some embodiments, the PBD conjugate is of Formula (XIVf), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVf).

In some embodiments, the PBD conjugate is of Formula (XIVg), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVg).

In some embodiments, the PBD conjugate is of Formula (XIVh), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVh).

In some embodiments, the PBD conjugate is of Formula (XIVi), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVi).

In some embodiments, the PBD conjugate is of Formula (XIVj), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVj).

In some embodiments, the PBD conjugate is of Formula (XIVk), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVk).

In some embodiments, the PBD conjugate is of Formula (XIVl), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVl).

In some embodiments, the PBD conjugate is of Formula (XIVm), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVm).

In some embodiments, the PBD conjugate is of Formula (XIVn), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVn).

In some embodiments, the PBD conjugate is of Formula (XIVo), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVo).

In some embodiments, the PBD conjugate is of Formula (XIVp), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVp).

In some embodiments, the PBD conjugate is of Formula (XIVq), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVq).

In some embodiments, the PBD conjugate is of Formula (XIVr), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVr).

In some embodiments, the PBD conjugate is of Formula (XIVs), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVs).

In some embodiments, the PBD conjugate is of Formula (XIVt), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVt).

In some embodiments, the PBD conjugate is of Formula (XIVu), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVu).

In some embodiments, the PBD conjugate is of Formula (XIVv), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVv).

In some embodiments, the PBD conjugate is of Formula (XIVw), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVw).

In some embodiments, the PBD conjugate is of Formula (XIVx), a tautomerthereof, a pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.

In some embodiments, the PBD conjugate is of Formula (XIVx).

In some embodiment the PBD drug moiety (D) of the PBD conjugate exhibitsbystander killing effects. In these embodiments the PBD drug moiety ishighly membrane-permeable whereas its hydrolysis products has a lowlevel of permeability and is locked in the cell.

In some embodiments, the PBD drug moiety (D) of the PBD conjugate is nota subtract for P-gp efflux pumps.

Pharmaceutical Compositions

Also included are pharmaceutical compositions comprising one or moreconjugates as disclosed herein in an acceptable carrier, such as astabilizer, buffer, and the like. The conjugates can be administered andintroduced into a subject by standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral administration including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion orintracranial, e.g., intrathecal or intraventricular, administration. Theconjugates can be formulated and used as sterile solutions and/orsuspensions for injectable administration; lyophilized powders forreconstitution prior to injection/infusion; topical compositions; astablets, capsules, or elixirs for oral administration; or suppositoriesfor rectal administration, and the other compositions known in the art.

The pharmaceutical compositions of the conjugates described herein canbe included in a container, pack, or dispenser together withinstructions for administration.

In some embodiments, the compositions can also contain more than oneactive compound as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Alternatively, or in addition, thecomposition can comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

In some embodiments, the active compounds (e.g., conjugates or drugs ofthe disclosure) are administered in combination therapy, i.e., combinedwith other agents, e.g., therapeutic agents, that are useful fortreating pathological conditions or disorders, such as various forms ofcancer, autoimmune disorders and inflammatory diseases. The term “incombination” in this context means that the agents are givensubstantially contemporaneously, either simultaneously or sequentially.If given sequentially, at the onset of administration of the secondcompound, the first of the two compounds is preferably still detectableat effective concentrations at the site of treatment.

In some embodiments, the combination therapy can include one or moreconjugates disclosed herein coformulated with, and/or coadministeredwith, one or more additional antibodies, which can be the same as theantibody used to form the conjugate or a different antibody.

In some embodiments, the combination therapy can include one or moretherapeutic agent and/or adjuvant. In certain embodiments, theadditional therapeutic agent is a small molecule inhibitor, anotherantibody-based therapy, a polypeptide or peptide-based therapy, anucleic acid-based therapy and/or other biologic.

In certain embodiments, the additional therapeutic agent is a cytotoxicagent, a chemotherapeutic agent, a growth inhibitory agent, anangiogenesis inhibitor, a PARP (poly(ADP)-ribose polymerase) inhibitor,an alkylating agent, an anti-metabolite, an anti-microtubule agent, atopoisomerase inhibitor, a cytotoxic antibiotic, any other nucleic aciddamaging agent or an immune checkpoint inhibitor. In one embodiment, thetherapeutic agent used in the treatment of cancer, includes but is notlimited to, a platinum compound (e.g., cisplatin or carboplatin); ataxane (e.g., paclitaxel or docetaxel); a topoisomerase inhibitor (e.g.,irinotecan or topotecan); an anthracycline (e.g., doxorubicin(ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); an anti-metabolite(e.g., gemcitabine, pemetrexed); cyclophosphamide; vinorelbine(NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; an angiogenesisinhibitor (e.g., Bevacizumab (Avastin®)), thalidomide, TNP-470, plateletfactor 4, interferon or endostatin); a PARP inhibitor (e.g., Olaparib(Lynparza™)); an immune checkpoint inhibitor, such as for example, amonoclonal antibody that targets either PD-1 or PD-L ((Pembrolizumab(Keytruda®), atezolizumab (MPDL3280A) or Nivolumab (Opdivo®)) or CTA-4(Ipilimumab (Yervoy®), a kinase inhibitor (e.g., sorafenib orerlotinib), a proteasome inhibitor (e.g., bortezomib or carfilzomib), animmune modulating agent (e.g., lenalidomide or IL-2), a radiation agent,an ALK inhibitor (e.g. crizotinib (Xalkori), ceritinib (Zykadia),alectinib (Alecensa), dalantercept (ACE-041), brigatinib (AP26113),entrectinib (NMS-E628), PF-06463922 TSR-011, CEP-37440 and X-396) and/ora biosimilar thereof and/or combinations thereof. Other suitable agentsinclude an agent considered standard of care by those skilled in the artand/or a chemotherapeutic agent well known to those skilled in the art.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA-4. In some embodiments, the immune checkpoint inhibitor is anantibody against CTLA-4. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against CTLA-4. In other embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstCTLA-4. In one embodiment, the anti-CTLA-4 antibody blocks the bindingof CTLA-4 to CD80 (B7-1) and/or CD86 (B7-2) expressed on antigenpresenting cells. Exemplary antibodies against CTLA-4 include, but arenot limited to, Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab(also known as Yervoy®, MDX-010, BMS-734016 and MDX-101); anti-CTLA4Antibody, clone 9H10 from Millipore; Pfizer's tremelimumab (CP-675,206,ticilimumab); and anti-CTLA4 antibody clone BNI3 from Abcam.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibodydisclosed in any of the following patent publications (hereinincorporated by reference): WO 2001014424; WO 2004035607;US2005/0201994; EP 1212422 BI; WO2003086459; WO2012120125; WO2000037504;WO2009100140; WO200609649; WO2005092380; WO2007123737; WO2006029219;WO20100979597; WO200612168; and WO1997020574. Additional CTLA-4antibodies are described in U.S. Pat. Nos. 5,811,097, 5,855,887,6,051,227, and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014;and/or U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281,incorporated herein by reference). In some embodiments, the anti-CTLA-4antibody is for example, those disclosed in: WO 98/42752; U.S. Pat. Nos.6,682,736 and 6,207,156; Hurwitz et al, Proc. Natl. Acad. Sci. USA,95(17): 10067-10071 (1998); Camacho et al, J. Clin. Oncol., 22(145):Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al, Cancer Res.,58:5301-5304 (1998) (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand asdisclosed in WO1996040915.

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor ofCTLA-4 expression. In some embodiments, anti-CTLA4 RNAi molecules maytake the form of the molecules described by Mello and Fire in PCTPublication Nos. WO 1999/032619 and WO 2001/029058; U.S. PublicationNos. 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839,2005/0100913, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576,and 2008/055443; and/or U.S. Pat. Nos. 6,506,559, 7,282,564, 7,538,095,and 7,560,438 (incorporated herein by reference). In some instances, theanti-CTLA4 RNAi molecules take the form of double stranded RNAimolecules described by Tuschl in European Patent No. EP 1309726(incorporated herein by reference). In some instances, the anti-CTLA4RNAi molecules take the form of double stranded RNAi molecules describedby Tuschl in U.S. Pat. Nos. 7,056,704 and 7,078,196 (incorporated hereinby reference). In some embodiments, the CTLA4 inhibitor is an aptamerdescribed in PCT Publication No. WO2004081021.

Additionally, the anti-CTLA4 RNAi molecules of the present disclosuremay take the form be RNA molecules described by Crooke in U.S. Pat. Nos.5,898,031, 6,107,094, 7,432,249, and 7,432,250, and European ApplicationNo. EP 0928290 (incorporated herein by reference).

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L1. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against PD-L1. In one embodiment, the immune checkpointinhibitor reduces the expression or activity of one or more immunecheckpoint proteins, such as PD-L1. In another embodiment, the immunecheckpoint inhibitor reduces the interaction between PD-1 and PD-L1.Exemplary immune checkpoint inhibitors include antibodies (e.g., ananti-PD-L1 antibody), RNAi molecules (e.g., anti-PD-L1 RNAi), antisensemolecules (e.g., an anti-PD-L1 antisense RNA), dominant negativeproteins (e.g., a dominant negative PD-L1 protein), and small moleculeinhibitors. Antibodies include monoclonal antibodies, humanizedantibodies, deimmunized antibodies, and Ig fusion proteins. An exemplaryanti-PD-L1 antibody includes clone EH12. Exemplary antibodies againstPD-L1 include: Genentech's MPDL3280A (RG7446); Anti-mouse PD-L1 antibodyClone 10F.9G2 (Cat #BE0101) from BioXcell; anti-PD-L1 monoclonalantibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer'sSquibb; MSB0010718C; mouse anti-PD-L1 Clone 29E.2A3; and AstraZeneca'sMEDI4736. In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1antibody disclosed in any of the following patent publications (hereinincorporated by reference): WO2013079174; CN101104640; WO2010036959;WO2013056716; WO2007005874; WO2010089411; WO2010077634; WO2004004771;WO2006133396; WO201309906; US 20140294898; WO2013181634 or WO2012145493.

In some embodiments, the PD-L1 inhibitor is a nucleic acid inhibitor ofPD-L1 expression. In some embodiments, the PD-L1 inhibitor is disclosedin one of the following patent publications (incorporated herein byreference): WO2011127180 or WO2011000841. In some embodiments, the PD-L1inhibitor is rapamycin.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L2. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L2. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L2. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against PD-L2. In some embodiments, the immune checkpointinhibitor reduces the expression or activity of one or more immunecheckpoint proteins, such as PD-L2. In other embodiments, the immunecheckpoint inhibitor reduces the interaction between PD-1 and PD-L2.Exemplary immune checkpoint inhibitors include antibodies (e.g., ananti-PD-L2 antibody), RNAi molecules (e.g., an anti-PD-L2 RNAi),antisense molecules (e.g., an anti-PD-L2 antisense RNA), dominantnegative proteins (e.g., a dominant negative PD-L2 protein), and smallmolecule inhibitors. Antibodies include monoclonal antibodies, humanizedantibodies, deimmunized antibodies, and Ig fusion proteins.

In some embodiments, the PD-L2 inhibitor is GlaxoSmithKline's AMP-224(Amplimmune). In some embodiments, the PD-L2 inhibitor is rHIgM12B7.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-1. In other embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstPD-1. In some embodiments, the inhibitors of PD-1 biological activity(or its ligands) disclosed in U.S. Pat. Nos. 7,029,674; 6,808,710; orU.S. Patent Application Nos: 20050250106 and 20050159351 can be used inthe combinations provided herein. Exemplary antibodies against PD-1include: Anti-mouse PD-1 antibody Clone J43 (Cat #BE0033-2) fromBioXcell; Anti-mouse PD-1 antibody Clone RMP1-14 (Cat #BE0146) fromBioXcell; mouse anti-PD-1 antibody Clone EH12; Merck's MK-3475anti-mouse PD-1 antibody (Keytruda®, pembrolizumab, lambrolizumab,h409A1 1); and AnaptysBio's anti-PD-1 antibody, known as ANB011;antibody MDX-1 106 (ONO-4538); Bristol-Myers Squibb's human IgG4monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106);AstraZeneca's AMP-514, and AMP-224; and Pidilizumab (CT-011 or hBAT-1),CureTech Ltd.

Additional exemplary anti-PD-1 antibodies are described by Goldberg etal, Blood 1 10(1): 186-192 (2007), Thompson et al, Clin. Cancer Res.13(6): 1757-1761 (2007), and Korman et al, International Application No.PCT/JP2006/309606 (publication no. WO 2006/121168 A1), each of which areexpressly incorporated by reference herein. In some embodiments, theanti-PD-1 antibody is an anti-PD-1 antibody disclosed in any of thefollowing patent publications (herein incorporated by reference):WO014557; WO2011110604; WO2008156712; US2012023752; WO2011110621;WO2004072286; WO2004056875; WO20100036959; WO2010029434; WO201213548;WO2002078731; WO2012145493; WO2010089411; WO2001014557; WO2013022091;WO2013019906; WO2003011911; US20140294898; and WO2010001617.

In some embodiments, the PD-1 inhibitor is a PD-1 binding protein asdisclosed in WO200914335 (herein incorporated by reference).

In some embodiments, the PD-1 inhibitor is a peptidomimetic inhibitor ofPD-1 as disclosed in WO2013132317 (herein incorporated by reference).

In some embodiments, the PD-1 inhibitor is an anti-mouse PD-1 mAb: cloneJ43, BioXCell (West Lebanon, N.H.).

In some embodiments, the PD-1 inhibitor is a PD-L1 protein, a PD-L2protein, or fragments, as well as antibody MDX-1 106 (ONO-4538) testedin clinical studies for the treatment of certain malignancies (Brahmeret al., J Clin Oncol. 2010 28(19): 3167-75, Epub 2010 Jun. 1). Otherblocking antibodies may be readily identified and prepared by theskilled person based on the known domain of interaction between PD-1 andPD-L1/PD-L2, as discussed above. In some embodiments, a peptidecorresponding to the IgV region of PD-1 or PD-L1/PD-L2 (or to a portionof this region) could be used as an antigen to develop blockingantibodies using methods well known in the art.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofIDO1. In some embodiments, the immune checkpoint inhibitor is a smallmolecule against IDOL. Exemplary small molecules against IDO1 include:Incyte's INCB024360, NSC-721782 (also known as 1-methyl-D-tryptophan),and Bristol Meyers Squibb's F001287.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofLAG3 (CD223). In some embodiments, the immune checkpoint inhibitor is anantibody against LAG3. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against LAG3. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against LAG3. In additional embodiments, an antibody againstLAG3 blocks the interaction of LAG3 with major histocompatibilitycomplex (MHC) class II molecules. Exemplary antibodies against LAG3include: anti-Lag-3 antibody clone eBioC9B7W (C₉B7W) from eBioscience;anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences; IMP321 (ImmuFact)from Immutep; anti-Lag3 antibody BMS-986016; and the LAG-3 chimericantibody A9H12. In some embodiments, the anti-LAG3 antibody is ananti-LAG3 antibody disclosed in any of the following patent publications(herein incorporated by reference): WO2010019570; WO2008132601; orWO2004078928.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst TIM3 (also known as HAVCR2). In some embodiments, the immunecheckpoint inhibitor is a monoclonal antibody against TIM3. In other oradditional embodiments, the immune checkpoint inhibitor is a human orhumanized antibody against TIM3. In additional embodiments, an antibodyagainst TIM3 blocks the interaction of TIM3 with galectin-9 (Gal9). Insome embodiments, the anti-TIM3 antibody is an anti-TIM3 antibodydisclosed in any of the following patent publications (hereinincorporated by reference): WO2013006490; WO201155607; WO2011159877; orWO200117057. In another embodiment, a TIM3 inhibitor is a TIM3 inhibitordisclosed in WO2009052623.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst B7-H3. In one embodiment, the immune checkpoint inhibitor isMGA271.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst MR. In one embodiment, the immune checkpoint inhibitor isLirilumab (IPH2101). In some embodiments, an antibody against MR blocksthe interaction of KIR with HLA.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD137 (also known as 4-1BB or TNFRSF9). In one embodiment, theimmune checkpoint inhibitor is urelumab (BMS-663513, Bristol-MyersSquibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592(Xencor). In one embodiment, an anti-CD137 antibody is an antibodydisclosed in U.S. Published Application No. US 2005/0095244: an antibodydisclosed in issued U.S. Pat. No. 7,288,638 (such as 20H4.9-IgG4 [1007or BMS-663513] or 20H4.9-IgG1 [BMS-663031]); an antibody disclosed inissued U.S. Pat. No. 6,887,673 [4E9 or BMS-554271]; an antibodydisclosed in issued U.S. Pat. No. 7,214,493; an antibody disclosed inissued U.S. Pat. No. 6,303,121; an antibody disclosed in issued U.S.Pat. No. 6,569,997; an antibody disclosed in issued U.S. Pat. No.6,905,685; an antibody disclosed in issued U.S. Pat. No. 6,355,476; anantibody disclosed in issued U.S. Pat. No. 6,362,325 [1D8 or BMS-469492;3H3 or BMS-469497; or 3E1]; an antibody disclosed in issued U.S. Pat.No. 6,974,863 (such as 53A2); or an antibody disclosed in issued U.S.Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1). In a further embodiment,the immune checkpoint inhibitor is one disclosed in WO 2014036412. Inanother embodiment, an antibody against CD137 blocks the interaction ofCD137 with CD137L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst PS. In one embodiment, the immune checkpoint inhibitor isBavituximab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD52. In one embodiment, the immune checkpoint inhibitor isalemtuzumab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD30. In one embodiment, the immune checkpoint inhibitor isbrentuximab vedotin. In another embodiment, an antibody against CD30blocks the interaction of CD30 with CD30L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD33. In one embodiment, the immune checkpoint inhibitor isgemtuzumab ozogamicin.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD20. In one embodiment, the immune checkpoint inhibitor isibritumomab tiuxetan. In another embodiment, the immune checkpointinhibitor is of atumumab. In another embodiment, the immune checkpointinhibitor is rituximab. In another embodiment, the immune checkpointinhibitor is tositumomab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD27 (also known as TNFRSF7). In one embodiment, the immunecheckpoint inhibitor is CDX-1127 (Celldex Therapeutics). In anotherembodiment, an antibody against CD27 blocks the interaction of CD27 withCD70.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst OX40 (also known as TNFRSF4 or CD134). In one embodiment, theimmune checkpoint inhibitor is anti-OX40 mouse IgG. In anotherembodiment, an antibody against 0×40 blocks the interaction of OX40 withOX40L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst glucocorticoid-induced tumor necrosis factor receptor (GITR). Inone embodiment, the immune checkpoint inhibitor is TRX518 (GITR, Inc.).In another embodiment, an antibody against GITR blocks the interactionof GITR with GITRL.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst inducible T-cell COStimulator (ICOS, also known as CD278). Inone embodiment, the immune checkpoint inhibitor is MEDI570 (MedImmune,LLC) or AMG557 (Amgen). In another embodiment, an antibody against ICOSblocks the interaction of ICOS with ICOSL and/or B7-H2.

In some embodiments, the immune checkpoint inhibitor is an inhibitoragainst BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2,or SLAM. As described elsewhere herein, an immune checkpoint inhibitorcan be one or more binding proteins, antibodies (or fragments orvariants thereof) that bind to immune checkpoint molecules, nucleicacids that downregulate expression of the immune checkpoint molecules,or any other molecules that bind to immune checkpoint molecules (i.e.small organic molecules, peptidomimetics, aptamers, etc.). In someinstances, an inhibitor of BTLA (CD272) is HVEM. In some instances, aninhibitor of CD160 is HVEM. In some cases, an inhibitor of 2B4 is CD48.In some instances, an inhibitor of LAIR1 is collagen. In some instances,an inhibitor of TIGHT is CD112, CD113, or CD155. In some instances, aninhibitor of CD28 is CD80 or CD86. In some instances, an inhibitor ofLIGHT is HVEM. In some instances, an inhibitor of DR3 is TL1A. In someinstances, an inhibitor of CD226 is CD155 or CD112. In some cases, aninhibitor of CD2 is CD48 or CD58. In some cases, SLAM is self-inhibitoryand an inhibitor of SLAM is SLAM.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that include, but are not limited to CTLA4 (cytotoxicT-lymphocyte antigen 4, also known as CD152), PD-L1 (programmed celldeath 1 ligand 1, also known as CD274), PDL2 programmed cell deathprotein 2), PD-1 (programmed cell death protein 1, also known as CD279),a B-7 family ligand (B7-H1, B7-H3, B7-H4) BTLA (B and T lymphocyteattenuator, also known as CD272), HVEM, TIM3 (T-cell membrane protein3), GAL9, LAG-3 (lymphocyte activation gene-3; CD223), VISTA, KIR(killer immunoglobulin receptor), 2B4 (also known as CD244), CD160,CGEN-15049, CHK1 (Checkpoint kinase 1), CHK2 (Checkpoint kinase 2), A2aR(adenosine A2a receptor), CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86,CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1 (indoleamine2,3-dioxygenase 1), IDO2 (indoleamine 2,3-dioxygenase 2), ICOS(inducible T cell costimulator), LAIR1, LIGHT (also known as TNFSF14, aTNF family member), MARCO (macrophage receptor with collagenousstructure), OX40 (also known as tumor necrosis factor receptorsuperfamily, member 4, TNFRSF4, and CD134) and its ligand OX40L (CD252),SLAM, TIGHT, VTCN1 or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor interacts with aligand of a checkpoint protein that comprises CTLA-4, PDL1, PDL2, PD1,BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1,CHK2, A2aR, a B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70,CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2,ICOS (inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophagereceptor with collagenous structure), OX-40, SLAM, TIGHT, VTCN1 or acombination thereof.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4, PDL1, PD1 or a combinationthereof.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4 and PD1 or a combinationthereof.

In certain embodiments, the immune checkpoint inhibitor comprisespembrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011),AMP-224, MDX-1 105, durvalumab (MED14736), MPDL3280A, BMS-936559,IPH2101, TSR-042, TSR-022, ipilimumab, lirilumab, atezolizumab,avelumab, tremelimumab, or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor is nivolumab(BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab,durvalumab, avelumab, or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor ispembrolizumab.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, inhaled, transdermal, or by injection/infusion. Such formsshould not prevent the composition or formulation from reaching a targetcell (i.e., a cell to which the drug is desirable for delivery). In someembodiments, pharmacological compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms that prevent the compositionor formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of the conjugate in the blood stream followed bydistribution throughout the entire body. Administration routes that leadto systemic absorption include, without limitation: intravenous,subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, andintramuscular. Each of these administration routes exposes theconjugates to an accessible diseased tissue. The rate of entry of anactive agent into the circulation has been shown to be a function ofmolecular weight or size. The use of a conjugate of this disclosure canlocalize the drug delivery in certain cells, such as cancer cells viathe specificity of PBRMs.

A “pharmaceutically acceptable formulation” means a composition orformulation that allows for the effective distribution of the conjugatesin the physical location most suitable for their desired activity. Inone embodiment, effective delivery occurs before clearance by thereticuloendothelial system or the production of off-target binding whichcan result in reduced efficacy or toxicity. Non-limiting examples ofagents suitable for formulation with the conjugates include:P-glycoprotein inhibitors (such as Pluronic P85), which can enhanceentry of active agents into the CNS; biodegradable polymers, such aspoly (DL-lactide-coglycolide) microspheres for sustained releasedelivery after intracerebral implantation; and loaded nanoparticles,such as those made of polybutylcyanoacrylate, which can deliver activeagents across the blood brain barrier and can alter neuronal uptakemechanisms.

Also included herein are pharmaceutical compositions prepared forstorage or administration, which include a pharmaceutically effectiveamount of the desired conjugates in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers, diluents, and/or excipients fortherapeutic use are well known in the pharmaceutical art. In someembodiments, buffers, preservatives, bulking agents, dispersants,stabilizers, dyes, can be provided. In addition, antioxidants andsuspending agents can be used Examples of suitable carriers, diluentsand/or excipients include, but are not limited to: (1) Dulbecco'sphosphate buffered saline, pH about 6.5, which would contain about 1mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl),and (3) 5% (w/v) dextrose.

The term “pharmaceutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Pharmaceutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to can be treated via gene silencing.

For any conjugate, the pharmaceutically effective amount can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. Therapeutic/prophylactic efficacy and toxicity may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

In some embodiments, a drug or its derivatives, drug-conjugates orPBRM-drug conjugates can be evaluated for their ability to inhibit tumorgrowth in several cell lines using Cell titer Glo. Dose response curvescan be generated using SoftMax Pro software and IC₅₀ values can bedetermined from four-parameter curve fitting. Cell lines employed caninclude those which are the targets of the PBRM and a control cell linethat is not the target of the PBRM contained in the test conjugates.

In one embodiment, the conjugates are formulated for parenteraladministration by injection including using conventional catheterizationtechniques or infusion. Formulations for injection may be presented inunit dosage form, e.g., in ampules or in multi-dose containers, with anadded preservative. The conjugates can be administered parenterally in asterile medium. The conjugate, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives, andbuffering agents can be dissolved in the vehicle. The term “parenteral”as used herein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like. In addition, there is provided a pharmaceuticalformulation comprising conjugates and a pharmaceutically acceptablecarrier. One or more of the conjugates can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and if desired other active ingredients.

The sterile injectable preparation can also be a sterile injectablesolution or suspension in a non-toxic parentally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, a bland fixed oil can be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The conjugates and compositions described herein may be administered inappropriate form, preferably parenterally, more preferablyintravenously. For parenteral administration, the conjugates orcompositions can be aqueous or nonaqueous sterile solutions, suspensionsor emulsions. Propylene glycol, vegetable oils and injectable organicesters, such as ethyl oleate, can be used as the solvent or vehicle. Thecompositions can also contain adjuvants, emulsifiers or dispersants.

Dosage levels of the order of from between about 0.001 mg and about 140mg per kilogram of body weight per day are useful in the treatment ofthe above-indicated conditions (between about 0.05 mg and about 7 g persubject per day). In some embodiments, the dosage administered to apatient is between about 0.001 mg/kg to about 100 mg/kg of the subject'sbody weight. In some embodiments, the dosage administered to a patientis between about 0.01 mg/kg to about 15 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered to a patient isbetween about 0.1 mg/kg and about 15 mg/kg of the subject's body weight.In some embodiments, the dosage administered to a patient is betweenabout 0.1 mg/kg and about 20 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 0.1 mg/kg to about5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered is between about 1mg/kg to about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 1 mg/kg to about10 mg/kg of the subject's body weight. The amount of conjugate that canbe combined with the carrier materials to produce a single dosage formvaries depending upon the host treated and the particular mode ofadministration. Dosage unit forms can generally contain from betweenabout 0.001 mg and about 100 mg; between about 0.01 mg and about 75 mg;or between about 0.01 mg and about 50 mg; or between about 0.01 mg andabout 25 mg; of a conjugate.

For intravenous administration, the dosage levels can comprise rangesdescribed in the preceding paragraphs, or from about 0.01 to about 200mg of a conjugate per kg of the animal's body weight. In one aspect, thecomposition can include from about 1 to about 100 mg of a conjugate perkg of the animal's body weight. In another aspect, the amountadministered will be in the range from about 0.1 to about 25 mg/kg ofbody weight of a compound.

In some embodiments, the conjugates can be administered are as follows.The conjugates can be given daily for about 5 days either as an i.v.,bolus each day for about 5 days, or as a continuous infusion for about 5days.

Alternatively, the conjugates can be administered once a week for sixweeks or longer. As another alternative, the conjugates can beadministered once every two or three weeks. Bolus doses are given inabout 50 to about 400 ml of normal saline to which about 5 to about 10ml of human serum albumin can be added. Continuous infusions are givenin about 250 to about 500 ml of normal saline, to which about 25 toabout 50 ml of human serum albumin can be added, per 24 hour period.

In some embodiments, about one to about four weeks after treatment, thepatient can receive a second course of treatment. Specific clinicalprotocols with regard to route of administration, excipients, diluents,dosages, and times can be determined by the skilled artisan as theclinical situation warrants.

In other embodiments, the therapeutically effective amount may beprovided on another regular schedule, i.e., daily, weekly, monthly, oryearly basis or on an irregular schedule with varying administrationdays, weeks, months, etc. Alternatively, the therapeutically effectiveamount to be administered may vary. In one embodiment, thetherapeutically effective amount for the first dose is higher than thetherapeutically effective amount for one or more of the subsequentdoses. In another embodiment, the therapeutically effective amount forthe first dose is lower than the therapeutically effective amount forone or more of the subsequent doses. Equivalent dosages may beadministered over various time periods including, but not limited to,about every 2 hours, about every 6 hours, about every 8 hours, aboutevery 12 hours, about every 24 hours, about every 36 hours, about every48 hours, about every 72 hours, about every week, about every two weeks,about every three weeks, about every month, and about every two months.The number and frequency of dosages corresponding to a completed courseof therapy will be determined according to the recommendations of therelevant regulatory bodies and judgment of a health-care practitioner.The therapeutically effective amounts described herein refer to totalamounts administered for a given time period; that is, if more than onedifferent conjugate described herein is administered, thetherapeutically effective amounts correspond to the total amountadministered. It is understood that the specific dose level for aparticular subject depends upon a variety of factors including theactivity of the specific conjugate, the age, body weight, generalhealth, sex, diet, time of administration, route of administration, andrate of excretion, combination with other active agents, and theseverity of the particular disease undergoing therapy.

In some embodiments, a therapeutically effective amount of a conjugatedisclosed herein relates generally to the amount needed to achieve atherapeutic objective. As noted above, this may be a binding interactionbetween the antibody and its target antigen that, in certain cases,interferes with the functioning of the target. The amount required to beadministered will furthermore depend on the binding affinity of theantibody for its specific antigen, and will also depend on the rate atwhich an administered antibody is depleted from the free volume othersubject to which it is administered. Common ranges for therapeuticallyeffective dosing of conjugates disclosed herein may be, by way ofnonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kgbody weight, from about 0.1 mg/kg body weight to about 100 mg/kg bodyweight or from about 0.1 mg/kg body weight to about 150 mg/kg bodyweight. Common dosing frequencies may range, for example, from twicedaily to once a month (e.g., once daily, once weekly; once every otherweek; once every 3 weeks or monthly). For example, conjugates disclosedherein can be administered (e.g., as a single dose weekly, every 2weeks, every 3 weeks, or monthly) at about 0.1 mg/kg to about 20 mg/kg(e.g., 0.2 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg,12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19mg/kg, or 20 mg/kg). For example, conjugates disclosed herein can beadministered (e.g., as a single dose weekly, every 2 weeks, every 3weeks, or monthly) at about 0.1 mg/kg to about 20 mg/kg (e.g., 0.2mg/kg, 0.5 mg/kg, 0.67 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 19mg/kg, or 20 mg/kg) for treating cancer.

For administration to non-human animals, the conjugates can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water so that the animal takes ina therapeutically appropriate quantity of the conjugates along with itsdiet. It can also be convenient to present the conjugates as a premixfor addition to the feed or drinking water.

The conjugates can also be administered to a subject in combination withother therapeutic compounds to increase the overall therapeutic effect.The use of multiple compounds to treat an indication can increase thebeneficial effects while reducing the presence of side effects. In someembodiments, the conjugates are used in combination withchemotherapeutic agents, such as those disclosed in U.S. Pat. No.7,303,749. In other embodiments the chemotherapeutic agents, include,but are not limited to letrozole, oxaliplatin, docetaxel, 5-FU,lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab,and gemcitabine.

The present disclosure also provides pharmaceutical kits comprising oneor more containers filled with one or more of the conjugates and/orcompositions of the present disclosure, including, one or morechemotherapeutic agents. Such kits can also include, for example, othercompounds and/or compositions, a device(s) for administering thecompounds and/or compositions, and written instructions in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products. The compositionsdescribed herein can be packaged as a single dose or for continuous orperiodic discontinuous administration. For continuous administration, apackage or kit can include the conjugates in each dosage unit (e.g.,solution or other unit described above or utilized in drug delivery),and optionally instructions for administering the doses daily, weekly,or monthly, for a predetermined length of time or as prescribed. Ifvarying concentrations of a composition, of the components of thecomposition, or the relative ratios of the conjugates or agents within acomposition over time is desired, a package or kit may contain asequence of dosage units which provide the desired variability.

A number of packages or kits are known in the art for dispensingpharmaceutical agents for periodic oral use. In one embodiment, thepackage has indicators for each period. In another embodiment, thepackage is a labeled blister package, dial dispenser package, or bottle.The packaging means of a kit may itself be geared for administration,such as a syringe, pipette, eye dropper, or other such apparatus, fromwhich the formulation may be applied to an affected area of the body,injected into a subject, or even applied to and mixed with the othercomponents of the kit.

Methods of Use

In some aspects, the present disclosure provides a method of treating asubject in need thereof (preferably mammals, most preferably humans andincludes males, females, infants, children and adults) by administeringa pharmaceutically effective amount of the conjugate (e.g., theantibody-drug conjugate (ADC)) of the present disclosure. In someembodiments, the conjugate (e.g., the antibody-drug conjugate (ADC)) ofthe present disclosure is administered in the form of soluble linearpolymers, copolymers, conjugates, colloids, particles, gels, soliditems, fibers, films, etc. Biodegradable biocompatible conjugates of thepresent disclosure can be used as drug carriers and drug carriercomponents, in systems of controlled drug release, preparations forlow-invasive surgical procedures, etc. Pharmaceutical formulations canbe injectable, implantable, etc.

In some aspects, the present disclosure provides a method of treating orpreventing a disease or disorder in a subject in need thereof,comprising administering to the subject a pharmaceutically effectiveamount of a conjugate (e.g., an antibody-drug conjugate (ADC)) of thepresent disclosure; wherein said conjugate releases one or more PBD drugmoieties upon biodegradation.

In some embodiments, the disease or disorder to be treated is ahyperproliferative disease, e.g., cancer.

In some embodiments, the conjugate (e.g., the antibody-drug conjugate(ADC)) of the present disclosure can be administered in vitro, in vitroand/or ex vivo to treat patients and/or to modulate the growth ofselected cell populations including, for example, cancer.

In some aspects, the present disclosure provides a method of treatingcancer, comprising administering to the subject a pharmaceuticallyeffective amount of a conjugate (e.g., an antibody-drug conjugate (ADC))of the present disclosure. In some embodiments, the particular types ofcancers that can be treated with the conjugates of the presentdisclosure include, but are not limited to: (1) solid tumors, includingbut not limited to fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma, multiforme astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, and retinoblastoma; (2) blood-bornecancers, including but not limited to acute lymphoblastic leukemia“ALL”, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cellleukemia, acute myeloblastic leukemia “AML”, acute promyelocyticleukemia “APL”, acute monoblastic leukemia, acute erythroleukemicleukemia, acute megakaryoblastic leukemia, acute myelomonocyticleukemia, acute nonlymphocyctic leukemia, acute undifferentiatedleukemia, chronic myelocytic leukemia “CML”, chronic lymphocyticleukemia “CLL”, hairy cell leukemia, multiple myeloma, acute and chronicleukemias, e.g., lymphoblastic myelogenous and lymphocytic myelocyticleukemias; and (3) lymphomas such as Hodgkin's disease, non-Hodgkin'sLymphoma, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

In some embodiments, the conjugate (e.g., the antibody-drug conjugate(ADC)) of the present disclosure can be administered in vitro, in vivoand/or ex vivo to treat autoimmune diseases.

In some aspects, the present disclosure provides a method of treating anautoimmune disease, comprising administering to the subject apharmaceutically effective amount of a conjugate (e.g., an antibody-drugconjugate (ADC)) of the present disclosure. In some embodiments, theautoimmune diseases that can be treated with the conjugates of thepresent disclosure include, but are not limited to, systemic lupus,rheumatoid arthritis, psoriasis, and multiple sclerosis; graftrejections, such as renal transplant rejection, liver transplantrejection, lung transplant rejection, cardiac transplant rejection, andbone marrow transplant rejection; graft versus host disease; viralinfections, such as CMV infection, HIV infection, and AIDS; and parasiteinfections, such as giardiasis, amoebiasis, schistosomiasis, and thelike.

In some aspects, the present disclosure provides a conjugate disclosedherein for use in the manufacture of a medicament useful for treating orlessening the severity of disorders, such as, characterized by abnormalgrowth of cells (e.g., cancer).

In some embodiments, the PBD drug moiety is locally delivered to aspecific target cell, tissue, or organ.

In some aspects, the present disclosure provides a method of treating adisease or disorder in a subject, comprising preparing an aqueousformulation of at least one conjugate of the present disclosure andparenterally injecting said formulation in the subject.

In some aspects, the present disclosure provides a method of treating adisease or disorder in a subject, comprising preparing an implantcomprising at least one conjugate of the present disclosure, andimplanting said implant into the subject. In some embodiments, theimplant is a biodegradable gel matrix.

In some aspects, the present disclosure provides a method for treatingof a subject in need thereof, comprising administering a conjugateaccording to the methods described above.

In some aspects, the present disclosure provides a method for elicitingan immune response in a subject, comprising administering a conjugate asin the methods described above.

In some aspects, the present disclosure provides a method of diagnosinga disease in a subject, comprising steps of:

administering a conjugate of the present disclosure, wherein theconjugate further comprises a detectable molecule; and

detecting the detectable molecule.

In some embodiments, the step of detecting the detectable molecule isperformed non-invasively. In some embodiments, the step of detecting thedetectable molecule is performed using suitable imaging equipment.

In some embodiments, the present disclosure provides a method fortreating an animal comprises administering to the animal a biodegradablebiocompatible conjugate of the present disclosure as a packing for asurgical wound from which a tumor or growth has been removed.

The biodegradable biocompatible conjugate packing will replace the tumorsite during recovery and degrade and dissipate as the wound heals.

In some embodiments, soluble or colloidal conjugates of the presentdisclosure are administered intravenously. In some embodiments, solubleor colloidal conjugates of the present disclosure are administered vialocal (e.g., subcutaneous, intramuscular) injection. In someembodiments, solid conjugates of the present disclosure (e.g.,particles, implants, drug delivery systems) are administered viaimplantation or injection.

In some embodiments, conjugates of the present disclosure comprising adetectable label are administered to study the patterns and dynamics oflabel distribution in animal body.

In some embodiments, the conjugate is associated with a diagnostic labelfor in vivo monitoring.

The conjugates described above can be used for therapeutic,preventative, and analytical (diagnostic) treatment of animals. Theconjugates are intended, generally, for parenteral administration, butin some cases may be administered by other routes.

In some embodiments, soluble or colloidal conjugates are administeredintravenously. In another embodiment, soluble or colloidal conjugatesare administered via local (e.g., subcutaneous, intramuscular)injection. In another embodiment, solid conjugates (e.g., particles,implants, drug delivery systems) are administered via implantation orinjection.

In another embodiment, conjugates comprising a detectable label areadministered to study the patterns and dynamics of label distribution inanimal body.

In some embodiments, any one or more of the conjugates disclosed hereinmay be used in practicing any of the methods described herein.

Diagnostic and Prophylactic Formulations

The PBD antibody conjugates disclosed herein are used in diagnostic andprophylactic formulations. In one embodiment, a PBD antibody conjugatedisclosed herein is administered to patients that are at risk ofdeveloping one or more of the aforementioned diseases, such as forexample, without limitation, cancer. A patient's or organ'spredisposition to one or more of the aforementioned indications can bedetermined using genotypic, serological or biochemical markers.

In another embodiment of the disclosure, a PBD antibody conjugatedisclosed herein is administered to human individuals diagnosed with aclinical indication associated with one or more of the aforementioneddiseases, such as for example, without limitation, cancer. Upondiagnosis, a PBD antibody conjugate disclosed herein is administered tomitigate or reverse the effects of the clinical indication associatedwith one or more of the aforementioned diseases. All methods describedherein can be performed in any suitable order unless otherwise indicatedherein or otherwise clearly contradicted by context. The use of any andall examples, or exemplary language (e.g., “such as”) provided herein,is intended merely to better illustrate the invention and is not to beconstrued as a limitation on the scope of the claims unless explicitlyotherwise claimed. No language in the specification is to be construedas indicating that any non-claimed element is essential to what isclaimed.

Definitions

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. In some embodiments,C₁-C₆ alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkylgroups. Examples of alkyl include, moieties having from one to sixcarbon atoms, such as, but not limited to, methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in another embodiment, a straight chain or branched alkylhas four or fewer carbon atoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g., fused,bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g.,C₃-C₁₀). Examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,1,2,3,4-tetrahydronaphthalenyl, and adamantyl. The term“heterocycloalkyl” refers to a saturated or unsaturated nonaromatic ringsystem having one or more heteroatoms (such as O, N, S, P, or Se) asring atoms, such as a 3-8 membered monocyclic, 7-12 membered bicyclic(fused, bridged, or spiro rings), or 11-14 membered tricyclic ringsystem (fused, bridged, or spiro rings) having, e.g., 1 or 1-2 or 1-3 or1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms,independently selected from the group consisting of nitrogen, oxygen andsulfur, unless specified otherwise. Examples of heterocycloalkyl groupsinclude, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl,dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl,azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl,tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl,tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl,1,4-dioxa-8-azaspiro[4.5]decanyl, 1,4-dioxaspiro[4.5]decanyl,1-oxaspiro[4.5]decanyl, 1-azaspiro[4.5]decanyl,3′H-spiro[cyclohexane-1,1′-isobenzofuran]-yl,7′H-spiro[cyclohexane-1,5′-furo[3,4-b]pyridin]-yl,3′H-spiro[cyclohexane-1,1′-furo[3,4-c]pyridin]-yl, and the like. In thecase of multicyclic non-aromatic rings, only one of the rings needs tobe non-aromatic (e.g., 1,2,3,4-tetrahydronaphthalenyl or2,3-dihydroindole). The terms “cycloalkylene” and “heterocycloalkylene”refer to the corresponding divalent groups, respectively.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, In some embodiments, alkyl, alkenyl, alkynyl,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

As used herein, “alkyl linker” or “alkylene linker” is intended toinclude C₁, C₂, C₃, C₄, C₅ or C₆ straight chain (linear or branched)saturated divalent aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. In some embodiments,C₁-C₆ alkylene linker is intended to include C₁, C₂, C₃, C₄, C₅ and C₆alkylene linker groups. Examples of alkylene linker include, moietieshaving from one to six carbon atoms, such as, but not limited to, methyl(—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. In some embodiments, the term “alkenyl” includesstraight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branchedalkenyl groups.

In certain embodiments, a straight chain or branched alkenyl group hassix or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straightchain, C₃-C₆ for branched chain). The term “C₂-C₆” includes alkenylgroups containing two to six carbon atoms. The term “C₃-C₆” includesalkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, In some embodiments, alkyl, alkenyl, alkynyl,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. In some embodiments, “alkynyl” includesstraight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl,pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branchedalkynyl groups. In certain embodiments, a straight chain or branchedalkynyl group has six or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkynyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkynyl groups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, In some embodiments, alkyl, alkenyl, alkynyl,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents. In some embodiments, substitutedheterocycloalkyl includes those substituted with one or more alkylgroups, such as 2,2,6,6-tetramethyl-piperidinyl and2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with one or more aromatic rings and do not containany heteroatom in the ring structure. Examples include phenyl,naphthalenyl, etc. The term “arylene” refers to the correspondingdivalent groups, such as phenylene.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable aromaticheterocyclic ring, such as a stable 5-, 6-, or 7-membered monocyclic or7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ringwhich consists of carbon atoms and one or more heteroatoms, e.g., 1 or1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or6 heteroatoms, independently selected from the group consisting ofnitrogen, oxygen and sulfur. The nitrogen atom may be substituted orunsubstituted (i.e., N or NR wherein R is H or other substituents, asdefined). The nitrogen and sulfur heteroatoms may optionally be oxidized(i.e., N→O and S(O)_(p), where p=1 or 2). It is to be noted that totalnumber of S and O atoms in the aromatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike. The term “heteroarylene” refers to the corresponding divalentgroups.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, quinoline, isoquinoline, naphthyridine, indole,benzofuran, purine, benzofuran, deazapurine, indolizine.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can besubstituted at one or more ring positions (e.g., the ring-forming carbonor heteroatom such as N) with such substituents as described above, Insome embodiments, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroarylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl such as benzo[d][1,3]dioxole-5-yl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. Carbocycle includes cycloalkyl and aryl. In someembodiments, a C₃-C₁₄ carbocycle is intended to include a monocyclic,bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or14 carbon atoms. Examples of carbocycles include, but are not limitedto, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are alsoincluded in the definition of carbocycle, including, In someembodiments, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, and [4.4.0]bicyclodecane and [2.2.2] bicyclooctane. A bridged ring occurs when oneor more carbon atoms link two non-adjacent carbon atoms. In someembodiments, bridge rings are one or two carbon atoms. It is noted thata bridge always converts a monocyclic ring into a tricyclic ring. When aring is bridged, the substituents recited for the ring may also bepresent on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) andspiro rings are also included.

As used herein, “heterocycle” or “heterocyclic group” includes any ringstructure (saturated, unsaturated, or aromatic) which contains at leastone ring heteroatom (e.g., 1-4 heteroatoms selected from N, O and S).Heterocycle includes heterocycloalkyl and heteroaryl. Examples ofheterocycles include, but are not limited to, morpholine, pyrrolidine,tetrahydrothiophene, piperidine, piperazine, oxetane, pyran,tetrahydropyran, azetidine, and tetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl (e.g.,benzo[d][1,3]dioxole-5-yl), morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R) occurs more than one time in any constituentor formula for a compound, its definition at each occurrence isindependent of its definition at every other occurrence. Thus, In someembodiments, if a group is shown to be substituted with 0-2 R moieties,then the group may optionally be substituted with up to two R moietiesand R at each occurrence is selected independently from the definitionof R. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

As used herein, the term “bis-oxy-alkylene” refers —O-alkylene-O—, inwhich alkylene can be linear or branched, e.g., —CH₂—, —CH(CH₃)₂—, or—(CH₂)₂—.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, In some embodiments, alkylgroups, alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. In someembodiments, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to —NH₂. “Alkylamino” includesgroups of compounds wherein the nitrogen of —NH₂ is bound to at leastone alkyl group. Examples of alkylamino groups include benzylamino,methylamino, ethylamino, phenethylamino, etc. “Dialkylamino” includesgroups wherein the nitrogen of —NH₂ is bound to two alkyl groups.Examples of dialkylamino groups include, but are not limited to,dimethylamino and diethylamino. “Arylamino” and “diarylamino” includegroups wherein the nitrogen is bound to at least one or two aryl groups,respectively. “Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxysubstituted with amino. “Alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. “Alkaminoalkyl” refers to analkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is alsobound to an alkyl group. “Acylamino” includes groups wherein nitrogen isbound to an acyl group. Examples of acylamino include, but are notlimited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureidogroups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

Compounds of the present disclosure that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.3-chloroperoxybenzoic acid (m-CPBA) and/or hydrogen peroxides) to affordother compounds of the present disclosure. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the presentdisclosure can be converted to N-hydroxy or N-alkoxy compounds. In someembodiments, N-hydroxy compounds can be prepared by oxidation of theparent amine by an oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent disclosure includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like, it being understood that not all isomers mayhave the same level of activity. In addition, a crystal polymorphism maybe present for the compounds represented by the formula. It is notedthat any crystal form, crystal form mixture, or anhydride or hydratethereof is included in the scope of the present disclosure.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present disclosure maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present disclosure, and the naming of the compounds doesnot exclude any isomeric forms, it being understood that not all isomersmay have the same level of activity.

Furthermore, the structures and other compounds discussed in thisdisclosure include all atropic isomers thereof, it being understood thatnot all atropic isomers may have the same level of activity. “Atropicisomers” are a type of stereoisomer in which the atoms of two isomersare arranged differently in space. Atropic isomers owe their existenceto a restricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertible by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine.

It is to be understood that the compounds of the present disclosure maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present disclosure, and the naming ofthe compounds does not exclude any tautomer form. It will be understoodthat certain tautomers may have a higher level of activity than others.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The compounds of any Formula described herein include the compoundsthemselves, as well as their salts, and their solvates, if applicable. Asalt, In some embodiments, can be formed between an anion and apositively charged group (e.g., amino) on a compound of the disclosure.Suitable anions include chloride, bromide, iodide, sulfate, bisulfate,sulfamate, nitrate, phosphate, citrate, methanesulfonate,trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate,succinate, fumarate, tartrate, tosylate, salicylate, lactate,naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term“pharmaceutically acceptable anion” refers to an anion suitable forforming a pharmaceutically acceptable salt. Likewise, a salt can also beformed between a cation and a negatively charged group (e.g.,carboxylate) on a compound of the disclosure. Suitable cations includesodium ion, potassium ion, magnesium ion, calcium ion, and an ammoniumcation such as tetramethylammonium ion. Examples of some suitablesubstituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. The compounds of thedisclosure also include those salts containing quaternary nitrogenatoms.

Examples of suitable inorganic anions include, but are not limited to,those derived from the following inorganic acids: hydrochloric,hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous,phosphoric, and phosphorous. Examples of suitable organic anionsinclude, but are not limited to, those derived from the followingorganic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic,hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic,lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic,oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic,propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric,toluenesulfonic, and valeric. Examples of suitable polymeric organicanions include, but are not limited to, those derived from the followingpolymeric acids: tannic acid, carboxymethyl cellulose.

Additionally, the compounds of the present disclosure, In someembodiments, the salts of the compounds, can exist in either hydrated orunhydrated (the anhydrous) form or as solvates with other solventmolecules. Non-limiting examples of hydrates include monohydrates,dihydrates, etc. Non-limiting examples of solvates include ethanolsolvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O. A hydrate refers to,In some embodiments, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of an active compound. Compounds of the disclosureinclude compounds where a nucleophilic solvent (H₂O, R^(A)OH, R^(A)NH₂,R^(A)SH) adds across the imine bond of the PBD moiety, which isillustrated below where the solvent is water or an alcohol (R^(A)OH,where R^(A) is an ether substituent as described above):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD. The balance of these equilibria depend on theconditions in which the compounds are found, as well as the nature ofthe moiety itself.

These compounds may be isolated in solid form, In some embodiments, bylyophilisation.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. In some embodiments, all of the compounds representedby Formula (I) are pyrrolo[2, 1-c][1, 4]benzodiazepines compounds(PBDs), and have Formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96,3147-3176,1996.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

The present disclosure provides methods for the synthesis of thecompounds of any of the Formulae and conjugates thereof describedherein. The present disclosure also provides detailed methods for thesynthesis of various disclosed conjugates of the present disclosureaccording to the following schemes as shown in the Examples.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the disclosure can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt thereof.

Compounds of the present disclosure can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001:Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,4^(th) Edition, Wiley-Interscience, 2007; R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995), incorporated by reference herein,are useful and recognized reference textbooks of organic synthesis knownto those in the art. The following descriptions of synthetic methods aredesigned to illustrate, but not to limit, general procedures for thepreparation of compounds of the present disclosure.

“Protein based recognition-molecule” or “PBRM” refers to a molecule thatrecognizes and binds to a cell surface marker or receptor such as, atransmembrane protein, surface immobilized protein, or proteoglycan.Examples of PBRMs include but are not limited to, antibodies (e.g.,Trastuzumab, Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab,Labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR2, EGFR, FGFR1, FGFR2,FGFR3, FGFR4, HER2, NaPi2b, c-Met, NOTCH1, NOTCH2, NOTCH3, NOTCH4,PD-L1, c-Kit, MUC1, MUC13 and anti-5T4) or peptides (LHRH receptortargeting peptides, EC-1 peptide), lipocalins, such as, In someembodiments, anticalins, proteins such as, In some embodiments,interferons, lymphokines, growth factors, colony stimulating factors,and the like, peptides or peptide mimics, and the like. The proteinbased recognition molecule, in addition to targeting the conjugate to aspecific cell, tissue or location, may also have certain therapeuticeffect such as antiproliferative (cytostatic and/or cytotoxic) activityagainst a target cell or pathway. The protein based recognition moleculecomprises or may be engineered to comprise at least one chemicallyreactive group such as, —COOH, primary amine, secondary amine —NHR, —SH,or a chemically reactive amino acid moiety or side chains such as, Insome embodiments, tyrosine, histidine, cysteine, or lysine. In someembodiments, a PBRM may be a ligand (LG) or targeting moiety whichspecifically binds or complexes with a cell surface molecule, such as acell surface receptor or antigen, for a given target cell population.Following specific binding or complexing of the ligand with itsreceptor, the cell is permissive for uptake of the ligand orligand-drug-conjugate, which is then internalized into the cell. As usedherein, a ligand that “specifically binds or complexes with” or“targets” a cell surface molecule preferentially associates with a cellsurface molecule via intermolecular forces. In some embodiments, theligand can preferentially associate with the cell surface molecule witha K_(d) of less than about 50 nM, less than about 5 nM, or less than 500pM. Techniques for measuring binding affinity of a ligand to a cellsurface molecule are well-known; In some embodiments, one suitabletechnique, is termed surface plasmon resonance (SPR). In someembodiments, the ligand is used for targeting and has no detectabletherapeutic effect as separate from the drug which it delivers. Inanother embodiment, the ligand functions both as a targeting moiety andas a therapeutic or immunomodulatory agent (e.g., to enhance theactivity of the active drug or prodrug).

Synthetic Methods

The conjugates of this disclosure having any of the Formulae describedherein may be prepared according to the procedures illustrated in Scheme1 and the Examples, from commercially available starting materials orstarting materials which can be prepared using literature procedures.

Any available techniques can be used to make the conjugates orcompositions thereof, and intermediates and components (e.g., scaffolds)useful for making them. For example, semi-synthetic and fully syntheticmethods may be used.

The general methods of producing the conjugates or scaffolds disclosedherein are illustrated in Scheme 1 below. More specific methods ofsyntheses of the conjugates are described in the Examples and for thescaffolds in application U.S. 62/572,010 filed Oct. 13, 2017. Thevariables (e.g., M^(P), M^(A), W^(D), L^(D), and L^(P′), etc.) in theseschemes have the same definitions as described herein unless otherwisespecified.

The synthetic processes of the disclosure can tolerate a wide variety offunctional groups; therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

PBD compounds used for the conjugates of the present disclosure can beprepared in a variety of ways using commercially available startingmaterials, compounds known in the literature, as described in co-pendingapplication U.S. Ser. No. 15/597,453 filed May 17, 2017, or from readilyprepared intermediates, by employing standard synthetic methods andprocedures either known to those skilled in the art, or which will beapparent to the skilled artisan in light of the teachings herein.Standard synthetic methods and procedures for the preparation of organicmolecules and functional group transformations and manipulations can beobtained from the relevant scientific literature or from standardtextbooks in the field. Although not limited to any one or severalsources, classic texts such as Smith, M. B., March, J., March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5^(th) edition,John Wiley & Sons: New York, 2001; and Greene, T. W., Wuts, P. G. M.,Protective Groups in Organic Synthesis, 3^(rd) edition, John Wiley &Sons: New York, 1999, incorporated by reference herein, are useful andrecognized reference textbooks of organic synthesis known to those inthe art. The following descriptions of synthetic methods are designed toillustrate, but not to limit, general procedures for the preparation ofcompounds of the present disclosure.

Conjugates of the present disclosure can be conveniently prepared by avariety of methods familiar to those skilled in the art. The conjugatesof the disclosure with each of the formulae described herein may beprepared according to the following procedures from commerciallyavailable starting materials or starting materials which can be preparedusing literature procedures. These procedures show the preparation ofrepresentative conjugates of this disclosure.

Conjugates designed, selected and/or optimized by methods describedabove, once produced, can be characterized using a variety of assaysknown to those skilled in the art to determine whether the conjugateshave biological activity. For example, the conjugates can becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen theconjugate molecules described herein for activity, using techniquesknown in the art. General methodologies for performing high-throughputscreening are described, for example, in Devlin (1998) High ThroughputScreening, Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughputassays can use one or more different assay techniques including, but notlimited to, those described below. Conjugates of the present disclosurecan also be prepared in a variety of ways using commercially availablestarting materials, compounds, antibodies, and antibody fragments eachof which are known in the literature, or from readily preparedintermediates, by employing standard synthetic methods and procedureseither known to those skilled in the art, or which will be apparent tothe skilled artisan in light of the teachings herein. In someembodiments, for the synthesis of conjugates of compounds of Formula(IV), where the antibody or antibody fragment is directly or indirectlylinked to the compound at position E″ or D″, methods and linkersdisclosed in WO2011/13063, WO2011/130616, WO2015/159076, WO2015/052535,WO2015/052534, WO2015/052321, WO2014/130879, WO2014/096365,WO2014/057122, WO2014/057073, WO2013/164593, WO2013/055993,WO2013/055990, WO2013/053873, WO2013/053871, WO2013/041606,WO2011/130616, and WO2011/130613 may be used. Each of these publicationsis incorporated herein by reference in its entirety.

As another example, for the synthesis of conjugates of compounds ofFormula (IV), where the antibody or antibody fragment is directly orindirectly linked to the compound at position R″₇, methods and linkersdisclosed in WO2014140174(A1) and WO2016/037644 may be used. Each ofthese publications is incorporated herein by reference in its entirety.

As another example, for the synthesis of conjugates of compounds ofFormula (IV), where the antibody or antibody fragment is directly orindirectly linked to the compound at position R″₁₀, methods and linkersdisclosed in WO 2013/055987, WO 2016/044560, WO 2016/044396,WO2015/159076, WO2015/095227, WO2015/095124, WO2015/052535,WO2015/052534, WO2015/052322, WO2014/174111, WO2014/096368,WO2014/057122, WO2014/057074, WO2014/022679, WO2014/011519,WO2014/011518, WO2013/177481, WO2013/055987, WO2011/130598, andWO2011/128650 may be used. Each of these publications is incorporatedherein by reference in its entirety.

Also included are pharmaceutical compositions comprising one or moreconjugates as disclosed herein in an acceptable carrier, such as astabilizer, buffer, and the like. The conjugates can be administered andintroduced into a subject by standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral administration including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion orintracranial, e.g., intrathecal or intraventricular, administration. Theconjugates can be formulated and used as sterile solutions and/orsuspensions for injectable administration; lyophilized powders forreconstitution prior to injection/infusion; topical compositions; astablets, capsules, or elixirs for oral administration; or suppositoriesfor rectal administration, and the other compositions known in the art.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, inhaled, transdermal, or by injection/infusion. Such formsshould not prevent the composition or formulation from reaching a targetcell (i.e., a cell to which the drug is desirable for delivery). In someembodiments, pharmacological compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms that prevent the compositionor formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of the modified polymer in the blood stream followed bydistribution throughout the entire body. Administration routes that leadto systemic absorption include, without limitation: intravenous,subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, andintramuscular. Each of these administration routes exposes the compoundor conjugate to an accessible diseased tissue. The rate of entry of anactive agent into the circulation has been shown to be a function ofmolecular weight or size. The use of a conjugate (e.g., an antibody-drugconjugate (ADC)) of this disclosure can localize the drug delivery incertain cells, such as cancer cells via the specificity of antibodies.

A “pharmaceutically acceptable formulation” means a composition orformulation that allows for the effective distribution of the conjugatesin the physical location most suitable for their desired activity. Insome embodiments, effective delivery occurs before clearance by thereticuloendothelial system or the production of off-target binding whichcan result in reduced efficacy or toxicity. Non-limiting examples ofagents suitable for formulation with the conjugates include:P-glycoprotein inhibitors (such as Pluronic P85), which can enhanceentry of active agents into the CNS; biodegradable polymers, such aspoly (DL-lactide-coglycolide) microspheres for sustained releasedelivery after intracerebral implantation; and loaded nanoparticles,such as those made of polybutylcyanoacrylate, which can deliver activeagents across the blood brain barrier and can alter neuronal uptakemechanisms.

Also included herein are pharmaceutical compositions prepared forstorage or administration, which include an effective amount of thedesired conjugates in a pharmaceutically acceptable carrier or diluent.Acceptable carriers, diluents, and/or excipients for therapeutic use arewell known in the pharmaceutical art. In some embodiments, buffers,preservatives, bulking agents, dispersants, stabilizers, dyes, can beprovided. In addition, antioxidants and suspending agents can be used.Examples of suitable carriers, diluents and/or excipients include, butare not limited to: (1) Dulbecco's phosphate buffered saline, pH about6.5, which would contain about 1 mg/ml to 25 mg/ml human serum albumin,(2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose.

The term “effective amount”, as used herein, refers to an amount of apharmaceutical agent to treat, ameliorate, or prevent an identifieddisease or condition, or to exhibit a detectable therapeutic orinhibitory effect. The effect can be detected by any assay or methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Effective amounts for a given situation canbe determined by routine experimentation that is within the skill andjudgment of the clinician. In a preferred aspect, the disease orcondition to can be treated via gene silencing.

For any conjugate, the effective amount can be estimated initiallyeither in cell culture assays, e.g., of neoplastic cells, or in animalmodels, usually rats, mice, rabbits, dogs, or pigs. The animal model mayalso be used to determine the appropriate concentration range and routeof administration. Such information can then be used to determine usefuldoses and routes for administration in humans. Therapeutic/prophylacticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose therapeutically effective in 50% of the population) and LD₅₀ (thedose lethal to 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀. Pharmaceutical compositions that exhibit largetherapeutic indices are preferred. The dosage may vary within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

In some embodiments, a drug or its derivatives, drug-polymer conjugatesor ADCs (including antibody-drug-polymer conjugates and antibody-drugconjugates) can be evaluated for their ability to inhibit tumor growthin several cell lines using CellTiter Glo®. Dose response curves can begenerated using SoftMax Pro software and IC₅₀ values can be determinedfrom four-parameter curve fitting. Cell lines employed can include thosewhich are the targets of the antibody and a control cell line that isnot the target of the antibody contained in the test conjugates.

In some embodiments, the PBD conjugates of the disclosure are formulatedfor parenteral administration by injection including using conventionalcatheterization techniques or infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The conjugates can beadministered parenterally in a sterile medium. The conjugate, dependingon the vehicle and concentration used, can either be suspended ordissolved in the vehicle. Advantageously, adjuvants such as localanesthetics, preservatives, and buffering agents can be dissolved in thevehicle. The term “parenteral” as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. One or moreof the conjugates can be present in association with one or morenon-toxic pharmaceutically acceptable carriers and/or diluents and/oradjuvants, and if desired other active ingredients.

The sterile injectable preparation can also be a sterile injectablesolution or suspension in a non-toxic parentally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, a bland fixed oil can be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The PBD conjugates and compositions described herein may be administeredin appropriate form, preferably parenterally, more preferablyintravenously. For parenteral administration, the compounds, conjugatesor compositions can be aqueous or nonaqueous sterile solutions,suspensions or emulsions. Propylene glycol, vegetable oils andinjectable organic esters, such as ethyl oleate, can be used as thesolvent or vehicle. The compositions can also contain adjuvants,emulsifiers or dispersants.

For PBD conjugates disclosed herein, the appropriate dosage levels willdepend on several factors, such as, In some embodiments, the type ofdisease to be treated, the severity and course of the disease, whetherthe compound is administered for preventing or therapeutic purposes,previous therapy, the patient's clinical history. Depending on the typeand severity of the disease, about 100 ng to about 25 mg (e.g., about 1μg/kg to 15 mg/kg, about 0.1-20 mg/kg) of the compound is an initialcandidate dosage for administration to the patient, whether, In someembodiments, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. An exemplarydosage of compound to be administered to a patient is in the range ofabout 0.1 to about 10 mg/kg of patient weight. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. An exemplary dosing regimen comprises a course ofadministering an initial loading dose of about 4 mg/kg, followed byadditional doses every week, two weeks, or three weeks of a compound.Other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays. Ranges disclosedherein are expressed as amount administered based on the subject'sweight, and one skilled in the art can easily express it as amountadministered per body surface area of the subject. In some embodiments,1 mg/kg body weight for a human adult is equivalent to about 37 mg/m²and 1 mg/kg body weight for a human child is equivalent to about 25mg/m².

For PBD conjugates disclosed herein, dosage levels of the order of frombetween about 0.01 mg and about 200 mg per kilogram of body weight perday are useful in the treatment of the target conditions (between about0.05 mg and about 7 g per subject per day). In some embodiments, thedosage administered to a patient is between about 0.01 mg/kg to about100 mg/kg of the subject's body weight. In some embodiments, the dosageadministered to a patient is between about 0.01 mg/kg to about 15 mg/kgof the subject's body weight. In some embodiments, the dosageadministered to a patient is between about 0.1 mg/kg and about 15 mg/kgof the subject's body weight. In some embodiments, the dosageadministered to a patient is between about 0.1 mg/kg and about 20 mg/kgof the subject's body weight. In some embodiments, the dosageadministered is between about 0.1 mg/kg to about 5 mg/kg or about 0.1mg/kg to about 10 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 1 mg/kg to about15 mg/kg of the subject's body weight. In some embodiments, the dosageadministered is between about 1 mg/kg to about 10 mg/kg of the subject'sbody weight.

The amount of conjugate that can be combined with the carrier materialsto produce a single dosage form varies depending upon the host treatedand the particular mode of administration. Dosage unit forms cangenerally contain from between about 0.01 mg and about 200 mg; between0.01 mg and about 150 mg; between 0.01 mg and about 100 mg; betweenabout 0.01 mg and about 75 mg; or between about 0.01 mg and about 50 mg;or between about 0.01 mg and about 25 mg; of a conjugate. In someembodiments, the PBD compound or conjugate of the disclosure can beadministered to a subject in need thereof (e.g., a human patient) at adose of about 100 mg, 3 times daily, or about 150 mg, 2 times daily, orabout 200 mg, 2 times daily, or about 50-70 mg, 3-4 times daily, orabout 100-125 mg, 2 times daily.

In some embodiments, the conjugates can be administered are as follows.The conjugates can be given daily for about 5 days either as an i.v.,bolus each day for about 5 days, or as a continuous infusion for about 5days.

Alternatively, the conjugates can be administered once a week for sixweeks or longer. As another alternative, the conjugates can beadministered once every two or three weeks. Bolus doses are given inabout 50 to about 400 ml of normal saline to which about 5 to about 10ml of human serum albumin can be added. Continuous infusions are givenin about 250 to about 500 ml of normal saline, to which about 25 toabout 50 ml of human serum albumin can be added, per 24 hour period.

In some embodiments about one to about four weeks after treatment, thepatient can receive a second course of treatment. Specific clinicalprotocols with regard to route of administration, excipients, diluents,dosages, and times can be determined by the skilled artisan as theclinical situation warrants.

It is understood that the specific dose level for a particular subjectdepends upon a variety of factors including the activity of the specificcompound or conjugate, the age, body weight, general health, sex, diet,time of administration, route of administration, and rate of excretion,combination with other active agents, and the severity of the particulardisease undergoing therapy.

For administration to non-human animals, the conjugates can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water so that the animal takes ina therapeutically appropriate quantity of the conjugates along with itsdiet. It can also be convenient to present the conjugates as a premixfor addition to the feed or drinking water.

The PBD conjugates disclosed herein can also be administered to asubject in combination with other therapeutic compounds to increase theoverall therapeutic effect. The use of multiple compounds to treat anindication can increase the beneficial effects while reducing thepresence of side effects. In some embodiments, the conjugates are usedin combination with chemotherapeutic agents, such as those disclosed inU.S. Pat. No. 7,303,749, U.S. 2016/0031887 and U.S. 2015/0133435, eachof which is herein incorporated by reference by its entirety. In otherembodiments, the chemotherapeutic agents, include, but are not limitedto letrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine,leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine.

The present disclosure also provides pharmaceutical kits comprising oneor more containers filled with one or more of the compounds, conjugatesand/or compositions of the present disclosure, including, one or morechemotherapeutic agents. Such kits can also include, In someembodiments, other compositions, a device(s) for administering thecompositions, and written instructions in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products.

In another aspect, the PBD conjugates of the disclosure are used inmethods of treating animals (preferably mammals, most preferably humansand includes males, females, infants, children and adults).

The conjugates of the disclosure may be used to provide a PBD conjugateat a target location.

The target location is preferably a proliferative cell population. Theantibody is an antibody for an antigen present in a proliferative cellpopulation.

In some embodiments, the antigen is absent or present at a reduced levelin a non-proliferative cell population compared to the amount of antigenpresent in the proliferative cell population, for example a tumor cellpopulation.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) of the disclosure include those withutility for anticancer activity. In particular, the ADC includes anantibody conjugated, i.e. covalently attached by a linker, to a PBDmoiety.

At the target location the linker may not be cleaved. The ADC of thedisclosure may have a cytotoxic effect without the cleavage of thelinker to release a PBD drug moiety. The ADC of the disclosureselectively deliver cytotoxic agent to tumor tissue whereby greaterselectivity, i.e., a lower efficacious dose, may be achieved.

In a further aspect, a conjugate as described herein is for use in thetreatment of a proliferative disease. A second aspect of the presentdisclosure provides the use of a conjugate compound in the manufactureof a medicament for treating a proliferative disease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. In some embodiments, assays which may convenientlybe used to assess the activity offered by a particular compound aredescribed in the examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumors (e.g. histiocytoma, glioma,astrocytoma, osteoma), cancers (e.g. lung cancer, small cell lungcancer, gastrointestinal cancer, bowel cancer, colon cancer, breastcarcinoma, ovarian carcinoma, prostate cancer, testicular cancer, livercancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer,sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias,psoriasis, bone diseases, fibroproliferative disorders (e.g. ofconnective tissues), and atherosclerosis. Cancers of particular interestinclude, but are not limited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

In some embodiments, the treatment is of a pancreatic cancer.

In some embodiments, the treatment is of a tumor having α_(v)β₆ integrinon the surface of the cell.

It is contemplated that the ADC of the present disclosure may be used totreat various diseases or disorders, e.g. characterized by theoverexpression of a tumor antigen. Exemplary conditions orhyperproliferative disorders include benign or malignant tumors;leukemia, hematological, and lymphoid malignancies. Others includeneuronal, glial, astrocytal, hypothalamic, glandular, macrophagal,epithelial, stromal, blastocoelic, inflammatory, angiogenic andimmunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, In some embodiments,rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLEand lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),osteoarthritis, autoimmune gastrointestinal and liver disorders (suchas, In some embodiments, inflammatory bowel diseases (e.g. ulcerativecolitis and Crohn's disease), autoimmune gastritis and perniciousanemia, autoimmune hepatitis, primary biliary cirrhosis, primarysclerosing cholangitis, and celiac disease), vasculitis (such as, Insome embodiments, ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and polyarteriitis), autoimmuneneurological disorders (such as, In some embodiments, multiplesclerosis, opsoclonus myoclonus syndrome, myasthenia gravis,neuromyelitis optica, Parkinson's disease, Alzheimer's disease, andautoimmune polyneuropathies), renal disorders (such as, In someembodiments, glomerulonephritis, Goodpasture syndrome, and Berger'sdisease), autoimmune dermatologic disorders (such as, In someembodiments, psoriasis, urticaria, hives, pemphigus vulgaris, bullouspemphigoid, and cutaneous lupus erythematosus), hematologic disorders(such as, In some embodiments, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, In some embodiments, inner ear disease and hearing loss),Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmuneendocrine disorders (such as, In some embodiments, diabetic-relatedautoimmune diseases such as insulin-dependent diabetes mellitus (IDDM),Addison's disease, and autoimmune thyroid disease (e.g. Graves' diseaseand thyroiditis)). More preferred such diseases include, In someembodiments, rheumatoid arthritis, ulcerative colitis, ANCA-associatedvasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves'disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, In some embodiments, theinhibition of the progress of the condition, and includes a reduction inthe rate of progress, a halt in the rate of progress, regression of thecondition, amelioration of the condition, and cure of the condition.Treatment as a prophylactic measure (i.e., prophylaxis, prevention) isalso included.

The subject/patient in need thereof may be an animal, mammal, aplacental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme(e.g., duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, arat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit),avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine(e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine(e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey(e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,In some embodiments, a fetus. In one preferred embodiment, thesubject/patient is a human.

In some embodiments, the patient is a population where each patient hasa tumor having αvβ6 integrin on the surface of the cell.

In certain embodiments, in practicing the method of the presentdisclosure, the conjugate further comprises or is associated with adiagnostic label. In certain exemplary embodiments, the diagnostic labelis selected from the group consisting of: radiopharmaceutical orradioactive isotopes for gamma scintigraphy and PET, contrast agent forMagnetic Resonance Imaging (MRI), contrast agent for computedtomography, contrast agent for X-ray imaging method, agent forultrasound diagnostic method, agent for neutron activation, moiety whichcan reflect, scatter or affect X-rays, ultrasounds, radiowaves andmicrowaves and fluorophores. In certain exemplary embodiments, theconjugate is further monitored in vivo.

Examples of diagnostic labels include, but are not limited to,diagnostic radiopharmaceutical or radioactive isotopes for gammascintigraphy and PET, contrast agent for Magnetic Resonance Imaging(MRI) (for example paramagnetic atoms and superparamagneticnanocrystals), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, and moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves, fluorophores in variousoptical procedures, etc. Diagnostic radiopharmaceuticals includeγ-emitting radionuclides, e.g., indium-111, technetium-99m andiodine-131, etc. Contrast agents for MRI (Magnetic Resonance Imaging)include magnetic compounds, e.g., paramagnetic ions, iron, manganese,gadolinium, lanthanides, organic paramagnetic moieties andsuperparamagnetic, ferromagnetic and antiferromagnetic compounds, e.g.,iron oxide colloids, ferrite colloids, etc. Contrast agents for computedtomography and other X-ray based imaging methods include compoundsabsorbing X-rays, e.g., iodine, barium, etc. Contrast agents forultrasound based methods include compounds which can absorb, reflect andscatter ultrasound waves, e.g., emulsions, crystals, gas bubbles, etc.Still other examples include substances useful for neutron activation,such as boron and gadolinium. Further, labels can be employed which canreflect, refract, scatter, or otherwise affect X-rays, ultrasound,radiowaves, microwaves and other rays useful in diagnostic procedures.Fluorescent labels can be used for photoimaging. In certain embodimentsa modifier comprises a paramagnetic ion or group.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following working examples are illustrative of the linkers, drugmolecules and antibodies or antibody fragments, and methods forpreparing same. These are not intended to be limiting and it will bereadily understood by one of skill in the art that other reagents ormethods may be utilized.

Abbreviations

The following abbreviations are used in the reaction schemes andsynthetic examples, which follow. This list is not meant to be anall-inclusive list of abbreviations used in the application asadditional standard abbreviations, which are readily understood by thoseskilled in the art of organic synthesis, can also be used in thesynthetic schemes and examples

-   -   ACN Acetonitrile    -   Alloc Allyloxycarbonyl    -   AcOH Acetic acid    -   DABCO 1,4-DIAZABICYCLO[2.2.2]OCTANE    -   DCHA 2-Methylindol-1-ylacetic acid    -   DCM Dichloromethane    -   EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline    -   EDCI        N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine    -   hydrochloride    -   EDTA Ethylenediaminetetraacetic acid    -   DIEA N,N-Diisopropylethylamine    -   DMA N,N-DIMETYLACETAMIDE    -   DMF Dimethylformamide    -   EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride    -   HOAt 1-Hydroxy-?-azabenzotriazole    -   HOBt Hydroxybenzotriazole    -   NHS 1-Hydroxypyrrolidine-2,5-dione (i.e., N-hydroxy-succinimide)    -   TEA Triethylamine    -   TEAA Triethylammonium acetate    -   TCEP Tris[2-carboxyethyl] phosphine    -   THF Tetrahydrofuran    -   MI Maleimide or maleimido    -   MTT 4-Methyltrityl    -   RP-HPLC Reverse-phase high performance liquid chromatography    -   SEC Size exclusion chromatography    -   WCX Weak cation exchange chromatography        General Information

Tumor growth inhibition (% TGI) was defined as the percent difference inmedian tumor volumes (MTVs) between treated and control groups.

Treatment efficacy was determined from the incidence and magnitude ofregression responses of the tumor size observed during the study.Treatment may cause partial regression (PR) or complete regression (CR)of the tumor in an animal. In a PR response, the tumor volume was 50% orless of its Day 1 volume for three consecutive measurements during thecourse of the study, and equal to or greater than 13.5 mm³ for one ormore of these three measurements. In a CR response, the tumor volume wasless than 13.5 mm3 for three consecutive measurements during the courseof the study. An animal with a CR response at the termination of a studywas additionally classified as a tumor-free survivor (TFS). Animals weremonitored for regression responses.

HPLC purification was performed on a Phenomenex Gemini 5 μm 110 Å,250×10 mm, 5 micron, semi-preparation column.

Whenever possible the drug content of the conjugates was determinedquantitatively by chromatography.

The protein content of the protein-drug conjugates was determinedspectrophotometrically at 280 nm or by ELISA.

Antibody-drug conjugates, can be purified (i.e., removal of residualunreacted drug, antibody, or starting materials) by extensivediafiltration. If necessary, additional purification by size exclusionchromatography can be conducted to remove any aggregated antibody-drugconjugates. In general, the antibody-drug conjugates as purifiedtypically contain <5% (e.g., <2% w/w) aggregated antibody-drugconjugates as determined by SEC; <0.5% (w/w) (e.g., <0.1% w/w) free(unconjugated) drug as determined by RP-HPLC or LC-MS/MS; <1% (w/w) offree drug conjugate as determined by SEC and/or RP-HPLC and <2% (w/w)(e.g., <1% w/w) unconjugated antibody or antibody fragment as determinedby HIC-HPLC and/or WCX HPLC. Reduced or partially reduced antibodieswere prepared using procedures described in the literature, see, forexample, Francisco et al., Blood 102 (4): 1458-1465 (2003). The totaldrug (conjugated and unconjugated) concentration was determined byRP-HPLC or back-calculation from DAR measured by CE-SDS.

RP-HPLC, or CE-SDS were used to characterize the specificity anddistribution of the cysteine bioconjugation sites in the PBRM-drugconjugates. The results gave the positional distribution of thedrug-conjugates on the heavy (H) and light (L) chains of the PBRM.

To determine the concentration of the free drug in a biological sample,an acidified sample was treated with acetonitrile. The free drug wasextracted and the acetonitrile supernatant was analyzed. To determinethe concentration of conjugated AF-HPA, the sample was subjected toexhaustive basic hydrolysis followed by immunocapture using anti-IgG1antibody magnetic beads. The acetonitrile supernatant containing thereleased AF-HPA and AF was analyzed RP-HPLC. The total antibody wasmeasured using the unique peptide after digestion. Analysis of free AFand AF-HPA was conducted by RP-HPLC using a C-4 column, an acetonitrilegradient and UV detection. Peak areas are integrated and compared to AFand AF-HPA standards. The method is quantitative for AF-HPA and AF inplasma and tissue homogenates and linear over the concentration rangesof 0.1 to 150 ng/mL. The total drug (AF-HPA) released after hydrolysiswith NaOH was measured under the same condition with the dynamic rangefrom 1 ng/mL to 5000 ng/mL. The total antibody standards range from 0.1μg/mL to 100 μg/mL.

General Procedure A: Partial selective reduction of protein (antibody)

The partial selective reduction of the inter-chain disulfide groups orunpaired disulfide in the relevant antibody prior to conjugation withthe polymer-drug conjugate is achieved by using a reducing agent, suchas, In some embodiments, TCEP, DTT or p-mercaptoethanol. When thereduction is performed with an excess of the reducing agent, thereducing agent is removed prior to conjugation by SEC. The degree ofconversion of the antibody disulfide groups into reactive sulfhydrylgroups depends on the stoichiometry of antibody, reducing agent, pH,temperature and/or duration of the reaction. When some but not all ofthe disulfide groups in the antibody are reduced, the reduced antibodyis a partially reduced antibody.

General Procedure B: Conjugation of Partially Reduced Antibody with DrugConjugate

The conjugation of the partially reduced antibody to the drug conjugateis conducted under neutral or slightly basic conditions (pH 6.5-8.5) atantibody concentrations of 1-10 mg/mL and drug conjugate concentrationsof 0.5-10 mg/mL. The drug conjugate is typically used in 1-5.0 foldexcess relative to the desired protein-drug conjugate stoichiometry.When the antibody is conjugated to the maleimido group of the drugconjugate, the conjugation is optionally terminated by the addition of awater-soluble maleimido blocking compound, such as, In some embodiments,N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine,2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid,mercaptomethanol (i.e., HOCH₂SH), benzyl thiol, and the like.

The resulting antibody-drug conjugate is typically purified bydiafiltration to remove any unconjugated polymer-drug conjugate,unconjugated drug and small molecule impurities. Alternatively oradditionally, appropriate chromatographic separation procedures such as,In some embodiments, size-exclusion chromatography, hydrophobicinteraction chromatography, ion chromatography such as, In someembodiments, WCX chromatography; reversed phase chromatography, hydroxylapatite chromatography, affinity chromatography or combinations thereofmay be used to purify the antibody-drug conjugate. The resultingpurified polymer-drug conjugate is typically formulated in a buffer atpH 5.0-6.5.

Other antibody-drug conjugates are synthesized with methods similar tothe procedure described herein, involving other antibodies and/orantibody fragments. Also antibody-drug conjugates with varying ratios ofdrug to antibody are obtained by varying the number of antibodysulfhydryl groups and drug load.

Example 1. Synthesis of Trastuzumab Conjugate 5

Part A:

To an ice-cold solution of compound 1 ((2.5 mg, 2.327 μmol, prepared asdescribed in U.S. 62/425,895) in water (80 μL) was added a solution of1-hydroxypyrrolidine-2,5-dione (NHS) (0.536 mg, 4.65 μmol) in water(20μ), then EDC (1.339 mg, 6.98 μmol) was added. The reaction mixturewas allowed to warm up to room temperature over 30 min. To this mixturewas added a solution of compound 2 (PC-5-129, prepared as described inU.S. Ser. No. 15/597,453) (2.82 mg, 3.14 μmol) in NMP/water (1:1, 40μL), followed by the addition of EDC (1.339 mg, 6.98 μmol). The reactionmixture was allowed to warm up to room temperature slowly. After about1.5 h, the crude product was purified by HPLC (10-70% acetonitrile/watercontaining 0.1% HOAc) to afford the desired Alloc-protected intermediate(2.5 mg, 1.3 μmol, 57% yield). ESI MS calc for C₈₆H₁₂₉N₁₇O₃₁ (M+2H)948.0; found 948.1. This purified Alloc-protected intermediate wasdissolved in CHCl₃ (150 μL) degassed by stirring at −78 C under highvacuum The reaction mixture was warmed to room temperature and treatedwith pyrrolidine (0.26 μmol) in CHCl₃ (10 μL), then with a solution ofPd(PPh₃)₄ (0.132 μmol) in CHCl₃, then allowed to stir at roomtemperature. After 1.5 h, the mixture diluted with acetonitrile (100μL), neutralized with HOAc (1 μL) and then purified by HPLC (C-18,10-70% acetonitrile/water containing 0.1% HOAc) to afford compound 3(1.5 mg, 0.828 μmol, 63% yield. ESI MS calc for (M+H) 1810.88; found1810.9.

Part B:

To a solution of compound 3 (2.2 mg, 0.377 μmol) in a mixture of NMP(125 μL)/DMSO (50 μL) and TEA (0.166 μL, 1.19 μmol) was added2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(1.0 mg, 2.4 μmol) in NMP (6.5 μL) at 0° C., and the resulting mixturewas stirred at room temperature. After 3 hours additional2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(2 equivalents) was added and the mixture was stirred overnight. Thereaction mixture was neutralized with acetic acid, diluted with waterand purified by HPLC (RP C18 column containing 0.1% HOAc (10-70% B over35 min) to afford compound 4 (1 mg, 40% yield). ESI MS calc forC₉₆H₁₄₃N₁₉O₃₅ (M+2H) 1061; found 1061.5.

Part C:

Conjugate 5 was prepared from Trastuzumab and compound 4 as described inU.S. Ser. No. 15/597,453. The purified conjugate had a PBD totrastuzumab ratio of 6.3 as determined by UV-Vis using molarextinction_(310 nm)=37,500 cm⁻¹M⁻¹ and 6280=25,394 cm⁻¹M⁻¹ for compound2 and ε_(280 nm)=226,107 cm⁻¹M⁻¹ for trastuzumab).

Example 2. Synthesis of Trastuzumab Conjugate 8

To a solution of compound 6 (88 mg, 0.078 mmol, prepared as described inU.S. 62/425,895) and compound 7 (60 mg, 0.060 mmol, prepared asdescribed in U.S. Ser. No. 15/597,453) in NMP (2 mL) was added NHS (9.8mg, 0.084 mmol), EDC (16 mg, 0.084 mmol), and DIEA (14.8 μL, 0.084 mmol)in succession. The mixture was stirred overnight at room temperaturethen purified by HPLC (0.1% formic acid in water:ACN, 10-90% B over 20min) and lyophilized to afford the desired intermediate maleimide as apale yellow solid (54.8 mg, 43% yield). ESI MS calc for C₉₇H₁₃₄N₂₂O₃(M+2H) 1051.5; found 1051.3. Compound 8 was prepared from thisintermediate maleimide using the procedure described in U.S. Ser. No.15/597,453. The purified conjugate 8 had a PBD to trastuzumab ratio of4.7 as determined by UV-Vis using molar extinction 322 m=38,072 cm⁻¹M⁻¹and 280=34,191 cm⁻¹M⁻¹ for compound 7 and C_(280 nm)=226,107 cm⁻¹M⁻¹ fortrastuzumab).

Example 3: Synthesis of Trastuzumab Conjugate 8A

Conjugate 8A was prepared as described in Example 2 and had a PBD totrastuzumab ratio of 4.7 as determined by UV-Vis using molar extinctionε_(322 nm)=38,072 cm⁻¹M⁻¹ and ε_(280 nm)=34,191 cm⁻¹M⁻¹ for compound 7and E280 n=226,107 cm⁻¹M⁻¹ for trastuzumab).

Example 4. Synthesis of Trastuzumab Conjugate 14

Part A:

To a solution of compound 9 (0.5 g, 2.64 mmol) and NaHCO₃ (0.222 g, 2.64mmol) in water (10 mL) was added a solution of allyl(2,5-dioxopyrrolidin-1-yl) carbonate (0.526 g, 2.64 mmol) in acetone (10mL). The mixture was then stirred overnight at room temperature. Acetonewas removed under vacuum and the pH was adjusted to 1-2 with 1 N HCl andthen extracted with EtOAc (2×30 mL). The organic phase was concentratedunder vacuum. The residue was suspended in acetonitrile and concentratedunder vacuum to afford compound 10 as a colorless solid (0.498 g). ESIMS: calc for C₁₀H₁₆N₃O₆ (M+H) 274.1; found 274.0.

Part B:

To a solution of compound 10 (13.23 mg, 0.048 mmol) and compound 11 (40mg, 0.048 mmol; prepared as described in U.S. Ser. No. 15/597,453) inTHF (1.0 mL) and DMA (0.2 mL) was added EEDQ (14.97 mg, 0.061 mmol) andthe mixture was stirred overnight. Additional DMA and EEDQ (2equivalents) was added and the mixture was stirred for another 20 h. Thecrude product was purified on silica gel (0-15% MeOH/DCM) to afford theAlloc-protected compound 11 intermediate (47 mg, 0.043 mmol, 90% yield).ESI MS: C₅₃H₅₆N₁₄O₁₂ (M+H) 1081.4; found 1080.8. This intermediate wasthen dissolved in a mixture DCM (1 mL) and DMF (0.5 mL) and treated withtriphenylphosphine (2.85 mg, 10.87 μmol) and pyrrolidine (4.49 μl, 0.054mmol) under argon. The resulting mixture was stirred at room temperaturefor 10 min, followed by the addition oftetrakis(triphenylphosphine)palladium(0) (2.51 mg, 2.174 μmol) and thesolution was stirred at room temperature for 20 min. The crude productwas then purified on silica gel (0-20% MeOH/DCM) to afford compound 12(26 mg, 0.026 mmol, 60.0% yield). ESI MS: calc for C₄₉H₅₃N₁₄O₁₀ (M+H)997.4; found 997.0.

Part C:

To a solution of compound 12 (26 mg, 0.026 mmol in DMF (2.0 ml) wasadded HOBt (3.88 mg, 0.029 mmol), 2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(12.20 mg, 0.029 mmol), triethylamine (4.0 μL, 0.029 mmol). The mixturewas stirred for 2 h at room temperature. The crude product was purifiedon HPLC (0.1% formic acid 10-90% ACN/water) to afford compound 13 (8.6mg, 6.58 μmol, 25.2 yield). ESI MS: calc for C₄₉H₅₃N₁₄O₁₀ (M+H) 1307.5;found 1306.8.

Part D:

Conjugate 14 was prepared from Trastuzumab and compound 13 as describedin Example 1. The purified conjugate had a PBD to trastuzumab ratio of3.0 as determined by UV-Vis using molar extinction ε_(322 nm)=38,072cm⁻¹M⁻¹ and 280 m=34,191 cm⁻¹M⁻¹ for compound 7 and 6280 m=226,107cm⁻¹M⁻¹ for trastuzumab).

Example 5. Synthesis of Trastuzumab Conjugate 22

Part A:

To an ice-cold mixture of (S)-tert-butyl2-amino-6-((tert-butoxycarbonyl)amino)hexanoate hydrochloride 15 (1.0 g,2.95 mmol) and sodium bicarbonate (0.545 g, 6.49 mmol) in a solution ofACN (10 mL) and water (15 mL) was added a solution of allyl(2,5-dioxopyrrolidin-1-yl) carbonate (0.646 g, 3.25 mmol) in ACN (5 mL).The ice bath was removed and the mixture was stirred overnight at roomtemperature. Acetonitrile was removed under vacuum and the reactionmixture was diluted with EtOAC (60 mL) and washed with brine. Theorganic phase was dried over MgSO₄, filtered and concentrated undervacuum. The crude product was purified on silica gel (Hex:EtOAc, 0-80%B) to afford compound 16 (1.09 g, 2.82 mmol, 96% yield). ESI MS: calcfor C₁₉H₃₄N₂NaO₆ (M+Na) 409.2; found 409.0.

Part B:

To an ice-cold solution of compound 16 (1.09 g, 2.82 mmol) in DCM (15mL) was added TFA (2.0 mL). The resulting solution was stirred at roomtemperature for 3 h, then the solvent was removed under vacuum and theresidue was placed under high vacuum overnight to yield compound 17 thatwas used in the next step without further purification (1.293 g, 2.82mmol, 100% yield). ESI MS: calc for C₁₀H₁₉N₂O₄ (M+H) 231.1; found 231.0.

Part C:

To a solution of compound 17 (1.293 g, 2.82 mmol) in DCM (15 mL) underargon at room temperature, was added triethylamine (2.163 mL, 15.52mmol). After 10 minutes, (chloro(p-tolyl)methylene)dibenzene (0.867 g,2.96 mmol) was added as a solid and stirred overnight under argon atroom temperature. The solvent was removed under vacuum, and the crudeproduct was purified on silica gel (0-30% MeOH in DCM) to affordcompound 18 (0.826 g, 1.697 mmol, 60.2% yield). ESI MS: calc. forC₃₀H₃₃N₂O₄ (M−H) 485.2; found 484.9.

Part D:

To a solution of compound 18 (29.5 mg, 0.061 mmol) and compound 11 (50mg, 0.061 mmol) in a mixture of THF (1.0 mL) and DMA (0.2 mL) was addedEEDQ (18.71 mg, 0.076 mmol) and the resulting mixture was stirredovernight. Additional DMA and EEDQ (0.2 equivalents) was added, and thereaction was stirred an additional 20 h. The crude product was purifiedon silica gel (0-15% MeOH/DCM) to afford compound 19 (47 mg, 0.036 mmol,60.0% yield). ESI MS: calc for C₇₃H₇₆N₁₃O₁₀ (M+H₂O+H) 1312.6; found1311.9.

Part E:

To a solution of compound 19 (47 mg, 0.036 mmol) in DCM (3 mL) was addedtriphenylphosphine (2.38 mg, 9.08 μmol) and pyrrolidine (3.75 μL, 0.045mmol) under argon and the mixture was stirred at room temperature for 10min. To this mixture was added tetrakis(triphenylphosphine)palladium(0)(2.098 mg, 1.815 μmol). The resulting solution was stirred at roomtemperature for 2 h, then the solvent was removed under vacuum and theresidue was purified on silica gel (0-20% MeOH/DCM) to afford compound20 (30 mg, 0.025 mmol, 68.3% yield). ESI MS: calc for C₆₉H₇₄N₃O₉ (M+NH₄)1227.6; found 1227.8.

Part F:

To a solution of compound 20 (30 mg, 0.025 mmol) in DMF (2.0 mL) wasadded HOBt (5.02 mg, 0.037 mmol), 2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(15.82 mg, 0.037 mmol), and triethylamine (5.18 μl, 0.037 mmol). Themixture was stirred 16 h at room temperature. The crude product waspurified by HPLC (0.1 Formic Acid, 10-90% ACN/Water) to afford thedesired MTT-protected maleimide intermediate (28 mg, 0.018 mmol, 74.3%yield). ESI MS: calc for C₈₃H₉₃N₆O₁₄ (M+NH₄) 1537.7; found 1537.8.

To a solution of the MTT-protected maleimide intermediate (28 mg, 0.018mmol) in DCM (1.4 mL) was added 2,2,2-trifluoroethanol (0.4 mL, 5.49mmol) and acetic acid (0.2 mL, 3.50 mmol) and the resulting mixture wasstirred at room temperature for 2 h. The crude product was purified onsilica gel (0-15% MeOH/DCM) to afford compound 21 (9.2 mg, 7.28 μmol,39.5% yield). ESI MS: calc for C₆₃H₇₄N₁₅O₁₄ (M+H) 1264.5; found 1264.7.

Part G:

Compound 22 was prepared from Trastuzumab and compound 21 as describedin Example 2. The purified conjugate had a PBD to trastuzumab ratio of3.6 as determined by UV-Vis using molar extinction 322 m==38,072 cm⁻¹M⁻¹and ε_(280 nm)=34,191 cm⁻¹M for compound 7 and ε_(280 nm)=226,107cm-‘M-’ for trastuzumab.

Example 6. Synthesis of Trastuzumab Conjugate 28

Compound 23 (1 g, 8.54 mmol) and NaHCO₃ (0.717 g, 8.54 mmol) weredissolved in acetone (42.7 mL) and water (42.7 mL). Allyl(2,5-dioxopyrrolidin-1-yl) carbonate (1.700 g, 8.54 mmol) was dissolvedin acetone (5 mL), and added to the reaction mixture, which was thenstirred for 12 h at room temperature. The reaction mixture was thenconcentrated under reduced pressure, acidified by the dropwise additionof concentrated HCl to pH 3, and extracted with EtOAc. The combinedorganic layers were washed with brine, dried over Na₂SO₄, andconcentrated to provide crude Alloc-Val-OH (1.85 g, 9.19 mmol, 108%yield) ESI MS: C₉H₁₄NO₄ ⁻ (M−H) 200.1; found 200.0 (negative mode),which was used in the next step without further purification.

To the crude Alloc-Val-OH (0.929 g, 4.62 mmol) was added HOAt (0.754 g,5.54 mmol), (S)-tert-butyl2-amino-6-((tert-butoxycarbonyl)amino)hexanoate hydrochloride (1.565 g,4.62 mmol), and EDCI (1.062 g, 5.54 mmol), under argon was adding Et₃N(3.22 mL, 23.08 mmol) and DMF (46.2 mL) and the resulting mixture wasstirred overnight at room temperature. The crude reaction mixture wasdiluted with water and extracted with DCM. The combined organic layerswere washed with brine and dried over Na₂SO₄, then concentrated andpurified on silica gel (100% DCM) to provide compound 24 (1.48 g, 3.05mmol, 66.0% yield) ESI MS: C₂₄H₄₃N₃NaO₇ ⁺ (M+Na) 508.3; found 508.0.

Part B:

To compound 24 (0.7152 g, 1.473 mmol) in DCM (2.95 mL), was added TFA(5.60 mL, 73.6 mmol) and the solution was stirred at room temperaturefor 6 hours. The reaction mixture was concentrated under reducedpressure to obtain the deprotected material as an oil (TFA salt), whichwas used directly in the next step. ESI MS: C₁₅H₂N₃O₅ ⁺ (M+H) 330.2;found 330.0.

The material from the previous step (485 mg, 1.473 mmol), in DMF (14.7mL) under argon at room temperature, was added Et₃N (1.13 mL, 8.10 mmolfollowed by the additional of (chloro(p-tolyl)methylene)dibenzene (453mg, 1.547 mmol) after 10 minutes and the reaction stirred at roomtemperature for 12 hours. The solvent was removed under reducedpressure, and the crude product was purified on silica gel (0-30% MeOHin DCM) to provide compound 25 (549.2 mg, 0.938 mmol, 63.7% yield). ESIMS: C₃₅H₄₂N₃O₅ ⁻ (M−H) 584.3; found 583.8.

Part C:

Compounds 11 (50 mg, 0.061 mmol, prepared as described in U.S. Ser. No.15/597,453), compound 25 (35.5 mg, 0.061 mmol) and EEDQ (19.46 mg, 0.079mmol) were dissolved in THF (908 μl) and DMF (303 μl) and stirred atroom temperature for 12 hours. The reaction mixture was concentrated,and the crude product was purified on silica gel (0-20% MeOH in DCM) toprovide the coupled product (37.3 mg, 0.027 mmol, 44.2% yield). ESI MS:C₇₈H₈₅N₁₄O₁₁ ⁺ (M+H) 1393.7; found 1393.7.

To the coupled product (37.3 mg, 0.027 mmol) were added DABCO (15.01 mg,0.134 mmol), and Pd(PPh₃)₄ (3.09 mg, 2.68 μmol) and DCM (1.33 mL) andthe mixture was stirred for 20 minutes at room temperature beforepurification on silica gel (0-40% MeOH in DCM) to provide compound 26(6.8 mg, 5.19 μmol, 19.40% yield). ESI MS: C₇₄H₈₁N₁₄O₉ ⁺ (M+H) 1309.6;found 1308.8

Part D:

Compound 26 (6.8 mg, 5.19 μmol) was dissolved in DMF (1 mL), and thentriethylamine (0.525 mg, 5.19 μmol) and 2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(2.209 mg, 5.19 μmol) were added and the mixture stirred at roomtemperature for 1 hour. The reaction mixture was concentrated on highvacuum and purified on silica gel (0-30% MeOH in DCM) to provide the MTTprotected intermediate (4.5 mg, 2.78 μmol, 53.5% yield). ESI MS:C₈₈H₉₉N₁₆O₁₅ ⁺ (M+H) 1619.8; found 1618.8.

The MTT protected intermediate (2.5 mg, 1.543 μmol) was dissolved in DCM(700 μl), 2,2,2-trifluoroethanol (200 μl, 2.74 mmol) and HOAc (100 μl,1.748 mmol) and stirred at room temperature for 1 hour. The concentratedreaction mixture was purified by reverse phase RP-HPLC (0.1% HOAc,10-100% ACN in H₂O), to obtain compound 27 (1.38 mg, 1.012 μmol, 65.6%yield). ESI MS: CO₈H₈₃N₁₆O₁₅ ⁺ (M+H) 1363.6; found 1363.8.

Part E:

Compound 28 was prepared from Trastuzumab and compound 27 as describedin Example 2. The purified conjugate had a PBD to trastuzumab ratio of3.9 as determined by UV-Vis using molar extinction ε_(322 nm)=38,072cm⁻¹M⁻¹ and ε_(280 nm)=34,191 cm⁻¹M⁻¹ for compound 7 andε_(280 nm)=226,107 cm⁻¹M⁻¹ for trastuzumab.

Example 7. Synthesis of Trastuzumab Conjugate 34

Part A:

To compound 29 (0.579 g, 2.3223 mmol, prepared from the DCHA salt) wasadded HOAt (0.379 g, 2.79 mmol), (S)-tert-butyl2-amino-6-((tert-butoxycarbonyl)amino)hexanoate hydrochloride (0.787 g,2.322 mmol), and EDCI (0.534 g, 2.79 mmol), under argon was added Et₃N(1.618 mL, 11.61 mmol) and DMF (23.22 mL) and the resulting mixture wasstirred for 12 hours at room temperature. The reaction mixture wasdiluted with water and extracted with DCM. The combined organic layerswere washed with brine, dried over Na₂SO₄, concentrated, and purified onsilica gel (0-30% MeOH in DCM) to provide compound 30 (1.239 g, 2.322mmol, 100% yield). ESI MS: C₂₈H₄₃N₃NaO₇ ⁺ (M+Na) 556.3; found 555.9.

Part B:

To compound 30 (558 mg, 1.046 mmol) in DCM (21 mL) at room temperature,and was added TFA (1192 μl, 15.68 mmol). After 6 hours, additional TFA(2 mL) was added. The reaction mixture was concentrated under reducedpressure to give the deprotected intermediate (395 mg, 1.046 mmol, 100%yield). ESI MS: C₁₉H₂₈N₃O₅ ⁺ (M+H) 378.2; found 378.0.

To the deprotected intermediate (0.395 g, 1.046 mmol), in DMF (10.46 mL)under argon at room temperature, was added Et₃N (0.802 mL, 5.75 mmol).After 10 minutes, (chloro(p-tolyl)methylene)dibenzene (0.322 g, 1.098mmol) was added and the reaction was stirred for 12 hours at roomtemperature, concentrated under reduced pressure, and the crude productwas purified on silica gel (0-30% MeOH in DCM) to provide compound 31(0.2845 g, 0.449 mmol, 42.9% yield). ESI MS: C₃₉H₄₂N₃O₅ ⁻ (M−H) 632.3;found 631.7 (M−H) negative mode.

Part C:

Compounds 11 (75 mg, 0.091 mmol, compound 31 (57.6 mg, 0.091 mmol), andEEDQ (29.2 mg, 0.118 mmol) dissolved in THF (1.36 mL) and DMF (454 μl)was stirred at room temperature for 12 hours. The reaction mixture wasconcentrated under reduced pressure, then purified on silica gel (0-20%MeOH in DCM) to provide the Alloc protected intermediate (66.5 mg, 0.046mmol, 50.8% yield). ESI MS: C₈₂H₈₅N₁₄O₁₁ ⁺ (M+H) 1441.7; found 1441.8.

To the Alloc protected intermediate (66.5 mg, 0.046 mmol),triphenylphosphine (3.02 mg, 0.012 mmol), pyrrolidine (4.74 μl, 0.058mmol) and DCM (1538 μl) under argon was added Pd(PPh₃)₄ (5.33 mg, 4.61μmol), and the resulting mixture was stirred at room temperature for 30min, followed by purification on silica gel (0-35% MeOH in DCM) toprovide compound 32 (62.6 mg, 0.046 mmol, 100% yield). ESI MS:C₇₈H₈₁N₁₄O₉ ⁺ (M+H) 1357.6; found 1357.7.

Part D:

To compound 32 (62.6 mg, 0.046 mmol) was added Et₃N (6.43 μl, 0.046mmol), DMF (922 μl) and 2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(19.62 mg, 0.046 mmol), and the mixture was stirred at room temperaturefor 15 minutes, concentrated on high vacuum, then purified on silica(0-35% MeOH in DCM) to provide the MTT protected intermediate (77 mg,0.046 mmol, 100% yield). ESI MS: C₉₂H₉₉N₁₆O₁₅ ⁺ (M+H) 1667.8; found1667.7.

To the MTT protected intermediate (77 mg, 0.046 mmol) was added DCM(2100 μl), 2,2,2-trifluoroethanol (600 μl, 8.23 mmol) and HOAc (300 μl,5.25 mmol) and the reaction mixture was stirred at room temperature for2 hours, concentrated under reduced pressure, then purified by RP-HPLC(10-100% ACN in H₂O with 0.1% HCO₂H) to provide 33 (4 mg, 2.83 μmol,6.14% yield). ESI MS: C₇₂H₈₃N₁₆O₁₅ ⁺ (M+H) 1411.6; found 1411.7.

Part E:

Compound 34 was prepared from Trastuzumab and compound 33 as describedin Example 2 The purified conjugate had a PBD to trastuzumab ratio of4.0 as determined by UV-Vis using molar extinction E322 m=38,072 cm⁻¹M⁻¹and 628) m=34,191 cm⁻¹M⁻¹ for compound 7 and ε_(280 nm)=226,107 cm⁻¹M⁻¹for trastuzumab.

Example 8. Synthesis of Trastuzumab Conjugate 35

Trastuzumab conjugate 35 was prepared as described in Example 2 exceptthat the corresponding PEG12 compound was used instead of the PEG8compound. The purified conjugate had a PBD to trastuzumab ratio of 5.2as determined by UV-Vis using molar extinction ε_(322 nm)=38,072 cm⁻¹M⁻¹and E280 m=34,191 cm⁻¹M⁻¹ for compound 7 and ε_(280 nm)=226,107 cm⁻¹M⁻¹for trastuzumab.

Example 9. Synthesis of Trastuzumab Conjugate 36

Conjugate 36 was prepared as described in US 2016/0082114A1 except thattrastuzumab was used. The purified conjugate 36 had a IGN to anti-Trop2antibody ratio of 2.3 as determined by UV-Vis using molar extinctionε_(330 nm)=15,484 cm⁻¹M⁻¹ and ε_(280 nm)=30,115 cm−1M−1 for IGN(according to WO2012/128868A1) and ε_(280 nm)=226,107 cm−1M−1 forTrastuzumab antibody).

Example 10. Cell Viability Assay for Antibody-Drug Conjugates

PBD conjugates were evaluated for their antiproliferation properties intumor cell lines in vitro using CellTiter-Glo® (Promega Corp). Cellswere plated in black walled 96-well plate and allowed to adhereovernight at 37° C. in a humidified atmosphere of 5% CO₂. BT474, SKBR3,NCI-N₈₇ cells (HER2 expressing cells), JIMT1 cells (HER2 mediumexpression level cells) and MCF7 cells (HER2 low expressing levelscells) and were plated at a density of 5,000 cells per well. The nextday the medium was replaced with 50 μL fresh medium and 50 μL of 2×stocks of PBD compounds or antibody-PBD conjugate were added toappropriate wells, mixed and incubated for 72 h. CellTiter-Glo® reagentwas added to the wells at room temperature and the luminescent signalwas measured after 10 min using a SpectraMax M5 plate reader (MolecularDevices). Dose response curves were generated using SoftMax Prosoftware. IC₅₀ values were determined from four-parameter curve fitting.

Table I gives illustrative results for the antiproliferation propertiesof the PBD and conjugates.

TABLE I BT474 SKBR3 N87 JIMT1 MCF7 Compound IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ No.(nmol/L) (nmol/L) (nmol/L) (nmol/L) (nmol/L)  8 0.21 0.03 0.08 96 43 8A0.06 0.01 0.04 300 253 14 300 0.048 0.260 300 300 22 2.45 0.020 0.11 300300 28 0.619 0.020 0.063 300 300 34 0.598 0.013 0.070 28.400 3.410 350.27 0.0184 0.0324 0.154 3

As shown in Tables I, the antibody-drug conjugates show efficacy in thetested cell lines.

Example 11. Tumor Growth Response to Administration of Antibody-DrugConjugates

Female CB-17 SCID mice were subcutaneously implanted with Calu-3 cells(n=10 for each group). Test compounds or vehicle were dosed IV as asingle dose on day 1. Tumor size was measured at the times indicated inFIG. 1 using digital calipers. Tumor volume was calculated and was usedto determine the delay in tumor growth. Tumor volumes are reported asthe mean±SEM for each group.

FIG. 1 provides the results for the tumor response in micesubcutaneously implanted with Calu-3 cells (n=10 for each group) afterIV administration of vehicle; and the Trastuzumab-drug conjugates:Example 4, Conjugate 14; Example 6, Conjugate 28; and Example 7,Conjugate 34; each at 1 mg/kg as a single dose at day 1; and Example 5,Conjugate 22, at 3 mg/kg as a single dose at day 1. The results showthat on day 90, Conjugate 22 resulted in 1 partial response; Conjugate28 in 5 partial responses and 2 complete responses; and Conjugate 34 in6 partial responses and 2 complete responses.

FIG. 2 provides the results for the tumor response in micesubcutaneously implanted with Calu-3 cells (n=10 for each group) afterIV administration of vehicle; and the Trastuzumab-drug conjugates:Example 2, Conjugate 8 at 3 mg/kg and 6 mg/kg each as a single dose atday 1; and Example 8, Conjugate 36 at 1 mg/kg and 3 mg/kg each as asingle dose at day 1. The results show that on day 102, Conjugate 8 at 3mg/kg resulted in 1 partial response, 9 complete responses and 9tumor-free survivals; and at 6 mg/kg resulted in 10 complete responsesand 10 tumor-free survivals; and Conjugate 36 at 3 mg/kg in 1 partialresponse.

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A conjugate of Formula (II):

or a pharmaceutically acceptable salt or solvate thereof, wherein: PBRMdenotes a protein based recognition-molecule; each occurrence of D isindependently a PBD drug moiety of Formula (IV),

a tautomer, pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer, wherein: E″is a direct or indirect linkage to the PBRM (e.g., antibody or antibodyfragment), E or

 in which

 denotes direct or indirect linkage to the PBRM via a functional groupof E; D″ is D′ or

 in which

 denotes direct or indirect linkage to the PBRM via a functional groupof D′; R″₇ is a direct or indirect linkage to the PBRM (e.g., antibodyor antibody fragment), R₇ or

 in which

 denotes direct or indirect linkage to the PBRM via a functional groupof R₇; R″₁₀ is a direct or indirect linkage to the PBRM, R₁₀ or

 in which

 denotes direct or indirect linkage the PBRM via a functional group ofR₁₀; and wherein the PBD drug moiety (D) is directly or indirectlylinked to the PBRM (e.g., antibody or antibody fragment) via afunctional group of one of E″, D″, R″₇, and R″₁₀, in which D′ is D1, D2,D3, or D4:

wherein the dotted line between C2 and C3 or between C2 and C1 in D1 orthe dotted line in D4 indicates the presence of a single or double bond;and m is 0, 1 or 2; when D′ is D1, the dotted line between C2 and C3 isa double bond, and m is 1, then R₁ is: (i) C₆₋₁₀ aryl group, optionallysubstituted by one or more substituents selected from —OH, halo, —NO₂,—CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, C₁₋₁₀ alkyl, C₃₋₁₀cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3- to 14-membered heterocycloalkyl, 5- to12-membered heteroaryl, bis-oxy-C₁₋₃ alkylene, —NR₁₃R₁₄, —S(═O)₂R₁₂,—S(═O)₂NR₁₃R₁₄, —SR₁₂, —SO_(x)M, —OSO_(x)M, —NR₉COR₁₉, —NH(C═NH)NH₂;(ii) C₁₋₅ alkyl; (iii) C₃₋₆ cycloalkyl;

 or (viii) halo; when D′ is D1, the dotted line between C2 and C3 is asingle bond, and m is 1, then R₁ is: (i) —OH, ═O, ═CH₂, —CN, —R₂, —OR₂,halo, ═CH—R₆, ═C(R₆)₂, —O—SO₂R₂, —CO₂R₂, —COR₂, —CHO, or —COOH; or

when D′ is D1 and m is 2, then each R₁ independently is halo and eitherboth R₁ are attached to the same carbon atom or one is attached to C2and the other is attached to C3; T is C₁₋₁₀ alkylene linker; A is

 wherein the —NH group of A is connected to the —C(O)-T- moiety ofFormula (IV) and the C═O moiety of A is connected to E; and each

 independently is

E is E1, E2, E3, E4, —OH, —NH—(C₁₋₆ alkylene)-R_(13a), —O—(CH₂)₃—NH₂,—O—CH(CH₃)—(CH₂)₂—NH₂ or —NH—(CH₂)₃—O—C(═O)—CH(CH₃)—NH₂:

 wherein the dotted line in E1 or E4 indicates the presence of a singleor double bond; each occurrence of R₂ and R₃ independently is anoptionally substituted C₁₋₈ alkyl, optionally substituted C₂₋₈ alkenyl,optionally substituted C₂₋₈ alkynyl, optionally substituted C₃₋₈cycloalkyl, optionally substituted 3- to 20-membered heterocycloalkyl,optionally substituted C₆₋₂₀ aryl or optionally substituted 5- to20-membered heteroaryl, and, optionally in relation to the group NR₂R₃,R₂ and R₃ together with the nitrogen atom to which they are attachedform an optionally substituted 4-, 5-, 6- or 7-membered heterocycloalkylor an optionally substituted 5- or 6-membered heteroaryl; R₄, R₅ and R₇are each independently —H, —R, —OH, —SH, —NHR₂_, —NR₂R₃, —NO₂, —SnMe₃,halo or a polyethylene glycol unit —(OCH₂CH₂)_(r)—OR_(a); or R₄ and R₇together form bis-oxy-C₁₋₃ alkylene; each R₆ independently is —H, —R₂,—CO₂R₂, —COR₂, —CHO, —CO₂H, or halo; each R₈ independently is —OH, halo,—NO₂, —CN, —N, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, —CONR₁₃R₁₄,—CO—NH—(C₁₋₆ alkylene)-R_(13a), C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycol unit—(OCH₂CH₂)_(r)—OR_(a), 3- to 14-membered heterocycloalkyl, 5- to12-membered heteroaryl, —S(═O)₂R₁₂, —S(═O)₂NR₁₃R₁₄, —SO_(x)M, —OSO_(x)M,—NR₉COR₁₉, —NH(C═NH)NH₂, —R₂₀—R₂₁—NR₁₃R₁₄,—R₂₀—R₂₁—NH—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃, or—O—P(O)(OH)—(OCH₂CH₂)_(n9)—OCH₃; each R₉ independently is C₁₋₁₀ alkyl,C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl; R¹⁰ is —H or anitrogen protecting group; R¹¹ is -QR^(Q) or -SO_(x)M, or R¹⁰ and R¹¹taken together with the nitrogen atom and carbon atom to which they arerespectively attached, form a N═C double bond; each R₁₂ independently isC₁₋₇ alkyl, 3- to 20-membered heterocycloalkyl, 5- to 20-memberedheteroaryl, or C₆₋₂₀ aryl; each occurrence of R₁₃ and R₁₄ are eachindependently H, C₁₋₁₀ alkyl, 3- to 20-membered heterocycloalkyl, 5- to20-membered heteroaryl, or C6-20 aryl; each R_(13a) independently is —OHor —NR₁₃R₁₄; R₁₅, R₁₆, R₁₇ and R₁₈ are each independently —H, —OH, halo,—NO₂, —CN, —N₃, —OR₂, —COOH, —COOR₂, —COR₂, —OCONR₁₃R₁₄, C₁₋₁₀ alkyl,C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, a polyethylene glycolunit —(OCH₂CH₂)_(r)—OR_(a), 3-14 membered heterocycloalkyl, 5- to12-membered heteroaryl, —NR₁₃R₁₄, —S(═O)₂R₁₂, —S(═O)₂NR₁₃R₁₄, —SR₁₂,—SO_(x)M, —OSO_(x)M, —NR₉COR₁₉ or —NH(C═NH)NH₂; each R₁₉ independentlyis C₁₋₁₀ alkyl, C₃₋₁₀ cycloalkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl; eachR₂₀ independently is a bond, C₆₋₁₀ arylene, 3-14 memberedheterocycloalkylene or 5- to 12-membered heteroarylene; each R₂₁independently is a bond or C₁₋₁₀ alkylene; R₃₁, R₃₂ and R₃₃ are eachindependently —H, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl or cyclopropyl,wherein the total number of carbon atoms in the R₁ group is no more than5; R₃₄ is —H, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, cyclopropyl, orphenyl wherein the phenyl is optionally substituted by one or more ofhalo, methyl, methoxy, pyridyl or thiophenyl; one of R_(35a) and R_(35b)is —H and the other is a phenyl group optionally substituted with one ormore of halo, methyl, methoxy, pyridyl or thiophenyl; R_(36a), R_(36b),R_(36c) are each independently —H or C₁₋₂ alkyl; R_(36d) is —OH, —SH,—COOH, —C(O)H, —N═C═O, —NHNH₂, —CONHNH₂,

 NHR^(N), wherein R^(N) is —H or C₁₋₄ alkyl; R_(37a) and R_(37b) areeach independently is —H, —F, C₁₋₄ alkyl, C₂₋₃ alkenyl, wherein thealkyl and alkenyl groups are optionally substituted by C₁₋₄ alkyl amidoor C₁₋₄ alkyl ester; or when one of R_(37a) and R_(37b) is —H, the otheris —CN or a C₁₋₄ alkyl ester; R₃₈ and R₃₉ are each independently H, R₁₃,═CH₂, ═CH—(CH₂)_(s1)—CH₃, ═O, (CH₂)_(s1)—OR₁₃, (CH₂)_(s1)—CO₂R₁₃,(CH₂)_(s1)—NR₁₃R₁₄, O—(CH₂)₂—NR₁₃R₁₄, NH—C(O)—R₁₃, O—(CH₂)s-NH—C(O)—R₁₃,O—(CH₂)s-C(O)NHR₁₃, (CH₂)_(s1)OS(═O)₂R₁₃, O—SO₂R₁₃, (CH₂)_(s1)—C(O)R₁₃and (CH₂)_(s1)—C(O)NR₁₃R₁₄; X₀ is CH₂, NR₆, C═O, BH, SO or SO₂; Y₀ is O,CH₂, NR₆ or S; Z₀ is absent or (CH₂)_(n); each X₁ independently isCR_(b), or N; each Y₁ independently is CH, NR_(a), O or S; each Z₁independently is CH, NR_(a), O or S; each R_(a) independently is H orC₁₋₄ alkyl; each R_(b) independently is H, OH, C₁₋₄ alkyl, or C₁₋₄alkoxyl; X₂ is CH, CH₂ or N; X₃ is CH or N; X₄ is NH, O or S; X₅ is NH,O or S; Q is O, S or NH; when Q is S or NH, then R^(Q) is —H oroptionally substituted C₁₋₂ alkyl; or when Q is O, then R^(Q) is —H oroptionally substituted C₁₋₂ alkyl, —SO_(x)M, —PO₃M,—(CH₂—CH₂—O)_(n9)CH₃, —(CH₂—CH₂O)_(n9)—(CH₂)₂—R₄₀,—C(O)—(CH₂—CH₂—O)_(n9)CH₃, —C(O)O—(CH₂—CH₂—O)_(n9)CH₃,—C(O)NH—)—(CH₂—CH₂—O)_(n9)CH₃,—(CH₂)_(n)—NH—C(O)—CH₂—O—CH₂—C(O)—NH—(CH₂—CH₂—O)_(n9)CH₃,—(CH₂)_(n)—NH—C(O)—(CH₂)_(n)—(CH₂—CH₂—O)_(n9)CH₃, a sugar moiety,

each M independently is H or a monovalent pharmaceutically acceptablecation; n is 1, 2 or 3; n₉ is 1, 2, 3, 4, 5, 6, 8, 12 or 24; each rindependently is an integer from 1 to 200; s is 1, 2, 3, 4, 5 or 6; s₁is 0, 1, 2, 3, 4, 5 or 6; t is 0, 1, or 2; R₄₀ is —SO₃H, —COOH,—C(O)NH(CH₂)₂SO₃H or —C(O)NH(CH₂)₂COOH; each x independently is 2 or 3;L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM; M^(P) is a Stretcher unit; a₁ is an integer from 0 to1; M^(A) comprises a peptide moiety that contains at least two aminoacids; T′ is a hydrophilic group and the

 between T′ and M^(A) denotes direct or indirect attachment of T′ andM^(A); each occurrence of L^(D) is independently a divalent linkermoiety connecting D to M^(A) and comprises at least one cleavable bondsuch that when the bond is broken, D is released in an active form forits intended therapeutic effect; and d₁₃ is an integer from 1 to
 20. 2.The conjugate of claim 1, wherein L^(P), when not connected to PBRM,comprises a terminal group W^(P), in which each W^(P) independently is:

wherein R^(1K) is a leaving group; R^(1A) is a sulfur protecting group;ring A is cycloalkyl or heterocycloalkyl; ring B is cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; R^(1J) is hydrogen or analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;R^(2J) is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclicmoiety; R^(3J) is C₁₋₆ alkyl; Z₁, Z₂, Z₃ and Z₇ are each independently acarbon or nitrogen atom; R^(4j) is hydrogen, halogen, OR, —NO₂, —CN,—S(O)₂R, C₁₋₂₄ alkyl (e.g., C₁₋₆ alkyl), or 6-24 membered aryl orheteroaryl, wherein the C1-24 alkyl (e.g., C₁₋₆ alkyl), or 6-24 memberedaryl or heteroaryl, is optionally substituted with one or more aryl orheteroaryl; or two R^(4j) together form an annelated cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; R is hydrogen, alkyl,heteroalkyl, cycloalkyl, or heterocycloalkyl R is hydrogen or analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;R^(5j) is C(R^(4j))₂, O, S or NR; and z₁ is an integer 1, 2, 3, 4, 5, 6,7, 8, 9 or
 10. 3. The conjugate of claim 1, wherein M^(P) is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M); R₃, R₅, R₁₇, and R₂₃ are as defined herein; R₄ is abond or —NR₅—(CR₂₀R₂₁)—C(O)—; each R₂₀ and R₂₁ independently ishydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, hydroxylated C₆₋₁₀ aryl,polyhydroxylated C₆₋₁₀ aryl, 5 to 12-membered heterocycle, C₃₋₈cycloalkyl, hydroxylated C₃₋₈ cycloalkyl, polyhydroxylated C₃₋₈cycloalkyl or a side chain of a natural or unnatural amino acid; each b₁independently is an integer from 0 to 6; e₁ is an integer from 0 to 8,each f₁ independently is an integer from 1 to 6; and g₂ is an integerfrom 1 to
 4. 4. The conjugate of claim 3, wherein M^(P), when present,is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M).
 5. The conjugate or scaffold of claim 1, whereinM^(A) comprises a peptide moiety that contains from three to about tenamino acids selected from glycine, serine, glutamic acid, aspartic acid,lysine, cysteine and a combination thereof.
 6. The conjugate of claim 1,wherein L^(D) comprises a peptide of 1 to 12 amino acids, wherein eachamino acid is independently selected from alanine, β-alanine, arginine,aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine,phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine,proline, tryptophan, valine, cysteine, methionine, selenocysteine,ornithine, penicillamine, aminoalkanoic acid, aminoalkynoic acid,aminoalkanedioic acid, aminobenzoic acid, amino-heterocyclo-alkanoicacid, heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoicacid, and derivatives thereof.
 7. The conjugate of claim 1, wherein thehydrophilic group comprises a polyalcohol or a derivative thereof, apolyether or a derivative thereof, or a combination thereof.
 8. Theconjugate of claim 1, wherein T′ comprises one or more of the followingfragments of the formula:

in which n₁ is an integer from 0 to about 6; each R₅₈ is independentlyhydrogen or C₁₋₈ alkyl; R₆₀ is a bond, a C₁₋₆ alkyl linker, or —CHR₅₉—in which R₅₉ is H, alkyl, cycloalkyl, or arylalkyl; R₆₁ is CH₂OR₆₂,COOR₆₂, —(CH₂)_(n2)COOR₆₂, or a heterocycloalkyl substituted with one ormore hydroxyl; R₆₂ is H or C₁₋₈ alkyl; and n₂ is an integer from 1 toabout
 5. 9. The conjugate of claim 8, wherein T′ comprises:


10. The conjugate of claim 1, wherein T′ comprises

in which n₄ is an integer from 1 to about 25; each R₆₃ is independentlyhydrogen or C₁₋₈ alkyl; R₆₄ is a bond or a C₁₋₈ alkyl linker; R₆₅ is H,C₁₋₈ alkyl, —(CH₂)_(n2)COOR₆₂, or —(CH₂)_(n2)COR₆₆; R₆₂ is H or C₁₋₈alkyl; R₆₆ is

 and n₂ is an integer from 1 to about
 5. 11. The conjugate of claim 1,wherein T′ comprises:

in which n₄ is an integer from about 2 to about
 20. 12. The conjugate ofclaim 1, wherein the PBD drug moiety (D) is of any one of formulae(V-1), (V-2), (V-3), (VII), (VII-1), (VII-2), (VII-3), (IX-a), (IX-b),(IX-c), (IX-d), (IX-e), (IX-f), (IX-g), (IX-h), (IX-i), (IX j), (IX-k),(IX-l), (IX-m), (IX-n), (IX-o), (IX-p), (IX-q), or (IX-r):

a tautomer, pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.
 13. Theconjugate of claim 1, wherein T is C₂₋₄ alkylene linker.
 14. Theconjugate of claim 1, wherein A is

wherein each X₁ independently is CH or N.
 15. The conjugate of claim 1,wherein E is

—OH, or —NH—(C₁₋₆ alkylene)-OH.
 16. The conjugate of claim 1, wherein

is

in which

denotes a direct or indirect linkage to the PBRM, L^(C), or L^(D), and

denotes a direct or indirect linkage to a remaining portion of D (e.g.,a direct or indirect linkage to A).
 17. The conjugate of claim 1,wherein the PBD drug moiety (D), prior to being connected to anotherportion of the conjugate, corresponds to a compound of any one ofFormula (XIIIa) to (XIIIm):

a tautomer, pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer.
 18. Theconjugate of claim 1, being a conjugate of Formula (XIVa) to (XIVx):

or a tautomer, pharmaceutically acceptable salt or solvate thereof, or apharmaceutically acceptable salt or solvate of the tautomer, wherein d₁₃is 3 to
 5. 19. A conjugate being selected from Formulae (XIVi), (XIVj)and (XIVo):

or a tautomer, pharmaceutically acceptable salt or solvates thereof, ora pharmaceutically acceptable salt or solvate of the tautomer.
 20. Apharmaceutical composition comprising the conjugate of claim 1 and apharmaceutically acceptable carrier.
 21. A method of treating a diseaseor disorder, comprising administering to a subject in need thereof apharmaceutically effective amount of the conjugate of claim 1, whereinthe disease or disorder is cancer.